Polynucleotides encoding brex system polypeptides and methods of using same

ABSTRACT

Isolated polynucleotides encoding a BREX system are provided. Accordingly there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same; Also provided is an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same. Also provided are compositions and methods for conferring phage resistance to bacteria or for conferring bacterial susceptibility to phages.

RELATED APPLICATION/S

This application claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application No. 61/894,993 filed Oct. 24, 2013, the contents of which are incorporated herein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to polynucleotides encoding BREX system polypeptides and methods of using same.

The ongoing arms race between bacteria and bacteriophages (phages) has led to the rapid evolution of efficient resistance systems to protect bacteria from phage infection (Stern and Sorek, 2011). These systems include restriction-modification systems enzymes that recognize and cleave foreign DNA (King and Murray, 1994), abortive infection (Abi) mechanisms that lead to the suicide of the infected host, thus protecting the colony against phage spread (Chopin et al., 2005), and the CRISPR/Cas adaptive defense system, which uses small RNAs to target and destroy invading phage DNA (Deveau et al., 2010). On the counter arm, as part of this continuous bacteria and phages arms race, successful phages had also developed numerous counter-resistance mechanisms to overcome bacterial defense (Stern and Sorek, 2011). Due to the rapid evolution and elaborated biological novelty associated with the bacteria-phage arms race, it is estimated that many additional, yet uncharacterized anti-phage defense systems are encoded by bacteria and archaea genomes (Stern and Sorek, 2011).

A broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Enormous amounts of bacteria are being cultivated each day in large fermentation vats, thus phage contamination can rapidly bring fermentations to a halt and cause economic setbacks, and is therefore considered a serious threat in these industries. The dairy fermentation industry has openly acknowledged the problem of phage and has been working with academia and starter culture companies to develop defense strategies and systems to curtail the propagation and evolution of phages for decades.

Anti-microbial phage therapy dates back to the early 1900s, after their co-discovery by Frederick Twort and Felix d'Hérelle (Twort F W 1915; and D'Hérelle 1917). Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted due to a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics (Miedzybrodzki R et al., 2012). Properly formulated and applied phages have sufficient potential to cure bacterial infections. The key advantage of phages as anti-microbial therapeutic agents is their potential to negatively impact only their specific bacterial targets. Other advantages include, for example, an increase in phage number over the course of treatment, tendency to only minimally disrupt normal flora, capability of disrupting bacterial biofilms, low inherent toxicities, and most importantly effectiveness against both antibiotic-sensitive and antibiotic-resistant bacteria.

In 1982, Chinenova and colleagues reported a unique phage defense phenotype in Streptomyces coelicolor A3(2), which was denoted Phage Growth Limitation (PGL) (Chinenova T. A. et al, 1982). In their work Chinenova et al. demonstrated that upon the first cycle of infection by the φ31 phage, Streptomyces coelicolor A3 was phage-sensitive and supported phage burst. However, phages emerging from this first cycle of infection could not successfully re-infect the Streptomyces coelicolor A3 host. Intriguingly, these phages were able to successfully infect strains of Streptomyces that do not carry the PGL system (Chinenova T. A. et al, 1982).

Further studies mapped the phenotype to a cluster of four genes, denoted pglW, pglX, pglY and pglZ, which were shown to reconstitute the above described PGL phenotype upon transfer to a PGL host (Sumby. P. & Smith, M. C. 2002). Of note, introduction of pglY and pglZ⁻ was not sufficient to confer a PGL+ phenotype in all mutants tested (Laity et al., 1993; Sumby et al. 2002). The domains encoded within these four genes do not resemble any classical combination of genes currently known to be involved in phage defense: pglZ is a member of the alkaline phosphatase superfamily; pglW has a serine/threonine kinase domain; pglX is an adenine-specific DNA methyltransferase; and pglY contains a p-loop ATPase domain (Sumby, P. & Smith, M. C. 2002). The PGL system described to date was not active against any other phage except for φC31 and its homoimmune relatives (Sumby. P. & Smith, M. C. 2002; Laity, C. et al. 1993).

A major characteristic of the PGL system described to date is the initial release of phage from the first infectious cycle followed by the attenuation of phage growth in the second. Various combinations of genes belonging to the PGL system, and predominantly pglZ, were found to be enriched within ‘defense islands’ (typical clustering of genes encoding defense system components in microbial genomes), providing additional support to the general involvement of these genes in a complex anti-phage defense system in multiple species (Makarova, K. S. et al. 2011; Makarova, K. S. et al. 2013). The discovery of the PGL system as an additional line of defense in bacteria may shed more light on the complex bacteria and phage arms race. However, a molecular mechanism that explains the activity of the PGL system has not yet been solved. Profound understanding of the molecular mechanism of this system might prove to be a powerful and economically important tool in molecular engineering applications (as was previously demonstrated with other complex phage resistance systems such as CRISPR-Cas).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.

According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.

According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a BREX system comprising:

-   -   (i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL;     -   (ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII;     -   (iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI;     -   (iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA;     -   (v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI; or     -   (vi) brxP, brxC/pglY, pglZ and brxL.

According to some embodiments of the invention, the nucleic acid construct comprising the polynucleotide encoding the BREX system further comprises a cis-acting regulatory element for directing expression of the nucleic acid sequence.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

According to an aspect of some embodiments of the present invention there is provided a phage defense composition, comprising as an active ingredient a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; and an acceptable carrier or diluent.

According to some embodiments of the invention, the nucleic acid construct or the composition comprises the BREX system formulated in a formulation suitable for cell penetration.

According to an aspect of some embodiments of the present invention there is provided an isolated cell genetically modified to express a BREX system selected from the group consisting of

(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW,

(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL,

(3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII,

(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI,

(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA,

(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or

(3vi) brxP, brxC/pglY, pglZ and brxL.

According to some embodiments of the invention, the genetically modified cell further being resistant to a first cycle phage infection.

According to some embodiments of the invention, the genetically modified cell being resistant to phage lysogeny.

According to some embodiments of the invention, the genetically modified cell being resistant to lytic phage.

According to some embodiments of the invention, the genetically modified cell being resistant to phage DNA replication.

According to an aspect of some embodiments of the present invention there is provided an isolated cell genetically modified to express a BREX system polypeptide selected from the group consisting of pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.

According to some embodiments of the invention, the isolated cell does not express a BREX system endogenously.

According to some embodiments of the invention, there is provided a method of protecting bacteria from phage attack, the method comprising expressing in the bacteria the isolated polynucleotide or the nucleic acid construct, thereby protecting the bacteria from phage attack.

According to an aspect of some embodiments of the present invention there is provided a method of protecting first bacteria from phage attack, the method comprising contacting the first bacteria with second bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, wherein the first bacteria and the second bacteria are non identical; thereby protecting the bacteria from phage attack.

According to some embodiments of the invention, the first bacteria does not express a BREX system endogenously.

According to some embodiments of the invention, the bacteria does not express a BREX system endogenously.

According to some embodiments of the invention, the phage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T and SPO2.

According to some embodiments of the invention, the phage is not Φ105, rho10 and rho14.

According to some embodiments of the invention, the phage is a lytic phage.

According to some embodiments of the invention, the lytic phage is SPO1 and/or SP82G.

According to an aspect of some embodiments of the present invention there is provided an isolated bacteria comprising a nucleic acid sequence encoding a BREX system and a transmissible genetic element expressing the BREX system, wherein the isolated bacteria do not endogenously express the BREX system and wherein the BREX system comprises brxC/pglY, pglZ and at least one of pglX, pglXL, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprises brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

According to some embodiments of the invention, the BREX system is type 1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.

According to some embodiments of the invention

-   -   (i) brxC/pglY is selected from the group of SEQ ID NO: 3-155,         157-765 and 767-1175,     -   (ii) pglZ is selected from the group of SEQ ID NO: 1176-1318,         1320-1856, 1858-2250, 6204 and 6205,     -   (iii) pglX is selected from the group of SEQ ID NO: 2251-3280         and 6186-6201.     -   (iv) pglXI is selected from the group of SEQ ID NO: 3281-3296,         3298-3356 and 3358-3403,     -   (v) brxP is selected from the group of SEQ ID NO: 3404-3440,     -   (vi) brxHI is selected from the group of SEQ ID NO: 3543-3642,     -   (vii) brxHII is selected from the group of SEQ ID NO: 3441-3460,         3462-3511 3513-3542 and 6173-6185,     -   (viii) brxL is selected from the group of SEQ ID NO: 3643-4412,         6165, 6166, 6169, 6170, 6202 and 6203.     -   (ix) brxD is selected from the group of SEQ ID NO: 4413-4488.     -   (x) brxA is selected from the group of SEQ ID NO: 4489-4621,         4623-5086, 5088-5415, 6167, 6168, 6171 and 6172,     -   (xi) brxB is selected from the group of SEQ ID NO: 5416-5947,         6206-6209,     -   (xii) brxF is selected from the group of SEQ ID NO: 5948-5957,         5959-5988 and 5990-6028.     -   (xiii) brxE is selected from the group of SEQ ID NO: 6029-6040,         and     -   (xiv) pglW is selected from the group of SEQ ID NO: 6041-6138.

According to an aspect of some embodiments of the present invention there is provided a method of inducing phage sensitivity in a bacterial cell, the method comprising contacting a bacterial cell which expresses a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducing sensitivity of the bacterial cell to phage infection.

According to some embodiments of the invention, the contacting is effected ex-vivo or in-vitro.

According to some embodiments of the invention, the contacting is effected in-vivo.

According to some embodiments of the invention, there is provided an isolated bacteria generated according to the method.

According to some embodiments of the invention, there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product the isolated polynucleotide, the construct, the composition, the isolated cell or the bacteria, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.

According to an aspect of some embodiments of the present invention there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product a bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.

According to some embodiments of the invention, the transmissible genetic element comprises a conjugative genetic element or mobilizable genetic element.

According to some embodiments of the invention, the food or feed is a dairy product.

According to some embodiments of the invention, the cell is a bacteria.

According to some embodiments of the invention, the bacteria is a species selected from the group consisting of Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, and Brevibacterium.

According to some embodiments of the invention, the bacteria is a lactic acid bacteria.

According to some embodiments of the invention, the bacteria is a species selected from the group consisting of Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species, Pediococcus species, Bifidobacterium, and Propionibacterium species.

According to some embodiments of the invention, there is provided a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising the isolated polynucleotide, the construct, the composition, the isolated cell or the isolated bacteria.

According to some embodiments of the invention, the product further comprises a dairy product.

According to an aspect of some embodiments of the present invention there is provided a method of treating a microbial infection in a subject in need thereof, the method comprising contacting the bacteria with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby treating the infection.

According to some embodiments of the invention, the method further comprising administering to the subject a phage therapy.

According to some embodiments of the invention, the method further comprising administering to the subject an antibiotic.

According to some embodiments of the invention, the method further comprising administering to the subject a phage therapy and/or an antibiotic.

According to an aspect of some embodiments of the present invention there is provided an article of manufacture identified for killing a bacteria comprising a packaging material packaging an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and a phage.

According to an aspect of some embodiments of the present invention there is provided an anti-microbial composition comprising as active ingredient an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and an acceptable carrier or diluent.

According to some embodiments of the invention, the composition further comprising a phage.

According to some embodiments of the invention, the anti BREX system agent is administered in a formulation suitable for cell penetration.

According to some embodiments of the invention, the anti BREX system agent is selected from the group consisting of a nucleic acid suitable for silencing expression, aptamers, small molecules and inhibitory peptides.

According to some embodiments of the invention, the anti BREX system agent is directed against pglX.

According to some embodiments of the invention, the anti BREX system agent is directed against brxC/pglY or pglZ. According to an aspect of some embodiments of the present invention there is provided a method of screening for identifying phage useful for infecting a bacteria, the method comprising:

(a) contacting a phage with a bacteria expressing BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW or comprising pglY, brxC/pglZ, pglX, pglW and at least one of brxD and brxHI;

(b) monitoring phage sensitivity of the bacteria, wherein an increase in phage sensitivity of the bacteria in the presence of the phage compared to phage sensitivity in the absence of the phage is indicative of a phage useful for infecting the bacteria.

According to some embodiments of the invention, the carrier is a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the BREX system is characterized by at least one of

(i) not being an abortive infection system;

(ii) not being a restriction modification system;

(iii) not preventing phage adsorption to a bacteria expressing same.

According to some embodiments of the invention, the pglX is a methylase.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-C demonstrate pglZ phylogeny and the classification of Bacteriophage Exclusion (BREX) subtypes. FIG. 1A is a phylogenetic tree representing pglZ protein occurrences. The tree is color coded according to the different BREX subtypes. Gene order and genomic organization of each BREX subtype is illustrated next to its relevant branch on the pglZ tree. Numbers depict bootstap values. FIG. 1B is a pie chart demonstrating the prevalence of the different BREX subtypes within the BREX superfamily system among the sequenced genomes that were analyzed. FIG. 1C shows representative appearances of the pglZ gene in a type 1 BREX six-gene cluster.

FIG. 2 shows local alignment between brxC from Bacillus cereus H3081.97 (type 1 BREX) and pglY from Streptomyces coelicolor A3(2) (type 2 BREX) demonstrating that the alignment between the two genes spans the P-loop domain [GXXXXGK(T/S), DUF2791, SEQ ID NO: 6162)]. Alignment coverage: 4%, e-value: 8e-09, identity: 24/57 (42%). Locus tags marked in parentheses.

FIG. 3 shows the structural alignment between brxA from Magnetospirillum magneticum (PDB entry 3BHW) and NusB from Aquifex aelicus (PDB entry 3R2C) demonstrating structural homology between brxA and NusB. BrxA is marked in red; NusB is marked in blue; NusE is marked in cyan; RNA is marked in grey. Alignment length: 44 amino acids.

FIGS. 4A-M demonstrate that integration of type 1 BREX system from Bacillus cereus H3081.97 into Bacillus subtilis BEST7003 strain confers resistance of the latter to phage infection. FIG. 4A depicts the type 1 BREX locus in Bacillus cereus H3081.97. Coordinates below the genes denote the position along the NZ_ABDLO1000007 contig in the draft genome of Bacillus cereus H3081.97. The orange box within brxC represents the position of the ATPase p-loop motif. FIG. 4B depicts the operon organization of the Bacillus cereus BREX system integrated in the Bacillus subtilis genome. FIGS. 4C-F are bivariate graphs indicating 12 hours culture dynamics of the control strain (BREX⁻, black) versus type 1 BREX-containing (red) strain of Bacillus subtilis following infection with different phages, as evaluated by optical density measurements in a 96-well plate format. Bacterial strains were exposed to phage at Time=0. Each graph represents 3 experiments with three technical triplicates for each biological replicate. Error bars represent SEM. FIG. 4C illustrates non-infected control (BREX⁻) and type 1 BREX-containing Bacillus subtilis cultures. FIGS. 4D-M illustrate culture dynamics of the BREX⁻ and type 1 BREX-containing Bacillus subtilis cultures following infection with Φ3T (FIG. 4D), rho10 (FIG. 4E), SP82G (FIG. 4F), SPO1 (FIG. 4G), 0105 (FIG. 4H). SPO2 (FIG. 4I), SP16 (FIG. 4J), Zeta (FIG. 4K), SPβ (FIG. 4L), and rho14 (FIG. 4M) phages.

FIGS. 5A-B are bivariate graphs demonstrating culture dynamics in the control strain (BREX⁻, black) versus type 1 BREX-containing (red) strain of Bacillus subtilis BEST7003 cultures following infection with different phages, as evaluated by optical density measurements. FIGS. 5A-B depict culture dynamics over an extended period (>30 hours) following infection with SOP1 (FIG. 5A) and SP28G (FIG. 5B) demonstrating temporally reproducible culture decline of the control BREX strain, but irreproducible culture decline at stochastic time points of the type 1 BREX⁺ strain. Re-growth following culture crash represents phage-resistant mutants. Each curve in the graphs represents a single technical replicate.

FIG. 6 is a bivariate graph demonstrating phage production during a one-step phage growth curve experiment with control strain (BREX⁻, black) versus type 1 BREX-containing (red) strain of Bacillus subtilis BEST7003 following infection with SPO1. The Y-axis represents absolute phage concentrations; the time points of maximal burst for BREX-lacking and BREX-containing strains are marked with black and red arrows, respectively. Error bars represent standard deviation of biological triplicates.

FIG. 7 demonstrates that BREX system confers resistance to phage first cycle of infection. Shown is a bivariate graph of phage production during a one-step phage growth curve experiment with control strain (BREX⁻, black) versus type 1 BREX-containing (red) strain of Bacillus subtilis BEST7003 infected with Φ3T. The Y-axis represents relative phage concentrations normalized to the value at the beginning of infection. Error bars represent standard deviation of biological triplicates.

FIG. 8 is a representative PCR photograph showing the presence of lysogens during a phage infection time course in the control Bacillus subtilis strain (BREX⁻, black), but not in type 1 BREX-containing Bacillus subtilis strain (BREX⁺, red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen-specific DNA are 293 bp, 485 bp, and 1218 bp, respectively.

FIG. 9 is a bivariate graph demonstrating culture dynamics in the control strain (BREX, black) versus type 1 BREX-containing (red) strain of Bacillus subtilis BEST7003 cultures following infection with increasing MOI of Φ3T phage showing that increasing the MOI shortens the time to culture crash for the BREX⁻ strain, but minimally influences the growth of the BREX⁺ strains. Error bars represent standard deviation of technical triplicates.

FIG. 10 is a bar graph representing the number of extracellular phages in the control strain (BREX⁻, black) versus type 1 BREX-containing (red) strain of Bacillus subtilis BEST7003 cultures 15 minutes following infection with phage Φ3T.

FIG. 11 is a bivariate graph illustrating that phage DNA replication is observed in control Bacillus subtilis BEST7003 strain (BREX, black) but does not occur in type 1 BREX-containing strain of Bacillus subtilis BEST7003 (BREX⁺, red). The Y-axis represents relative phage DNA concentration normalized to the value at the beginning of infection, as evaluated by Illumina sequencing of total cellular DNA.

FIG. 12 is a southern blot photograph demonstrating phage Φ3T genome at different time points following infection of control (BREX⁻, black) vs. type 1 BREX-containing (BREX⁺, red) Bacillus subtilis BEST7003 strains. Numbers marking each lane indicate the time (in minutes) following infection. U lanes indicate uninfected control.

FIGS. 13A-B demonstrate the methylation activity of BREX. FIG. 13A depicts the consensus sequence around the modified base in the type 1 BREX-containing Bacillus subtilis BEST7003 strain. The arrow marks the modified base. FIG. 13B is a table summarizing the statistics of the modified motifs in the type 1 BREX-containing Bacillus subtilis BEST7003 strain.

FIGS. 14A-B are bivariate graphs demonstrating culture dynamics of non-infected (FIG. 14A) or following infection with phage Φ3T (FIG. 14B) in the control strain (BREX⁻, black), type 1 BREX-containing (red) and type 1 BREX without pglX-containing (pglXΔ, green) strains of Bacillus subtilis BEST7003 cultures, as evaluated by optical density measurements.

FIG. 15 is a Common Tree of bacteria and archaea as represented in the NCBI Taxonomy resource demonstrating the Distribution of BREX systems across the phylogenetic tree of bacteria and archaea. Organisms in which a BREX system exists are colored following the BREX subtype color code from FIG. 1A. Extensive horizontal transfer is observed by the lack of coherence between the species tree and the pglZ phylogeny.

FIG. 16 is a phylogenetic tree representing brxC/pglY protein occurrences as determined by aligning only the P-loop domain showing that the brxC/pglY phylogeny follows the classification of BREX subtypes and co-evolves with pglZ. The tree is color coded according to the different BREX subtypes as in FIG. 1A. Only brxC/PglY proteins appearing in complete systems were taken for this analysis.

FIGS. 17A-C illustrate frequent irregularities in the adenine-specific methylase pglX in BREX type 1. FIG. 17A depicts irregular genotypes (duplication, inversion and premature stop codon) associated with pglX. FIG. 17B depicts genomic organization of BREX system type 1 in Methanobrevibacter smithii ATCC 35061 (NC_009515 SEQ ID NO: 6163). FIG. 17C depicts genomic organization of BREX system type 1 in Lactobacillus rhamnosus GG. A cassette switch between the short and the long forms of pglX is observed when the sequences of two isolates of Lactobacillus rhamnosus GG (accessions FM179322 (NC_013198 SEQ ID NO: 1) and AP011548 (NC_017482 SEQ ID NO: 2) respectively are compared. Repeat sequences between the short and long forms are in black.

FIG. 18 is an agarose gel photograph of DNA extracted from type 1 BREX-containing and BREX-lacking strains of Bacillus subtilis BEST7003 cultures, following time course infection with phage Φ3T demonstrating no degradation of host DNA.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to polynucleotides encoding BREX system polypeptides and methods of using same.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The continuous bacteria-phages arms race has led to rapid evolution of both anti-phage bacterial resistance systems and counter-resistance mechanisms developed by phages, many of which are yet uncharacterized. A broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Development of defense strategies and systems to curtail the propagation and evolution of phages in fermentation vats is therefore warranted. On the counter arm, properly formulated and applied phages have sufficient potential to cure bacterial infections addressing the therapeutic need for new antibiotics.

The PGL system has previously been reported as conferring phage resistance manifested by attenuation of phage growth in the second cycle.

Whilst reducing the present invention to practice, the present inventors have now uncovered a novel multi-gene phage resistance system broadly distributed in bacteria and archaea, which the present inventors denoted BREX (Bacteriophage Elusion) system. The newly discovered BREX system shares some structural and functional similarities with the previously described PGL system. The abundance of this system and the efficiency in which it protects against phages implies that it plays an important role as a major line of defense encoded by bacteria against phages.

Specifically, the present inventors have uncovered that BREX system confers complete or partial resistance against phages spanning a wide phylogeny of phage types, including lytic and temperate (also referred lysogenic) phages, even in the first cycle of infection. Alternatively, mutations (e.g.; frame shift in pglX) affecting the functionality of the BREX system abrogate phage resistance.

Taken together, the present teachings suggest that BREX system and functional portions thereof can be used for conferring phage resistance. Such naturally and engineered bacteria can be utilized for example in the dairy industry, where phages cause serious annual losses, as well as in other industries that rely on large-scale bacterial fermentation for biotechnological production. Alternatively, anti-BREX system agents can be used as antibiotics.

As is illustrated hereinunder and in the examples section which follows, the present inventors have uncovered that BREX system confers complete or partial resistance against phages spanning a wide phylogeny of phage types, including lytic and temperate phages, even in the first cycle of infection. Even more so, mutations (e.g.; frame shift in pglX) affecting the functionality of the BREX system abrogate phage resistance. Specifically, the present inventors have shown that the BREX system exists in almost 10% of sequenced microbial genomes, and can be divided into six coherent subtypes containing 4-8 genes each, two of which are core genes, pglZ and brxC/pglY, present in all subtypes (Examples 1-2, Tables 1-8, FIGS. 1A-C). As detailed in Example 2 and Tables 2-7, in most cases, a single BREX system per organism was found, and in several of the identified systems, one of the genes was either missing or has become a pseudogene. The inventors have further demonstrated that the BREX system undergoes extensive horizontal transfer, with subtype 1, the most frequent subtype of this system, possibly the ancestral form of BREX (Example 5, FIGS. 1A-C, 16.)

The inventors have further demonstrated (Examples 3 and 4. FIGS. 4A-M, 5A-D, 6, 7 and Table 9) that integration of the BREX type 1 system into Bacillus subtilis strain lacking an endogenous BREX system confers complete protection from infection by the temperate SPβ, SP16, Zeta, Φ3T, and SPO2 phages and partial protection from infection by the lytic SP82G and SPO1 phages, even in the first cycle of infection.

The present inventors have gained insight into BREX mechanism of action (Example 4, FIGS. 8-12), accordingly it is demonstrated that integration of the type 1 BREX system into Bacillus subtilis strain allows phage adsorption but prevents phage lysogeny and phage DNA replication. In addition, the system methylates the host chromosomal DNA at a specific motif while sparing the phage DNA and this methylation is likely to be essential for the system's activity. Specifically, the methylase gene pglX was essential for the function of the type 1 BREX system and presented high rates of irregularities (Example 4. FIGS. 13A-B and 14A-B and Example 6. FIGS. 17A-B), thus marking pglX as possibly undergoing frequent phase-variation, and suggesting that this gene might confer specificity in the BREX system, or, alternatively, is particularly toxic. The data strongly indicates that the system does not an act via the previously described PGL mechanism or via abortive infection (Abi) or simple restriction/modification mechanisms.

Consequently, the present invention provides methods and compositions for use in the food, feed, medical and veterinary industries to confer phage resistance. On the other hand, the present invention provides methods suitable for use in the food, feed, medical and veterinary industries to generate phage with broader host range that can be used for more effective bio-control of bacteria.

Thus, according to a first aspect of the present invention, there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.

According to a second aspect of the present invention, there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein the BREX system confers phage resistance to a bacteria recombinantly expressing same.

According to another aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a BREX system comprising:

(i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL;

(ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII;

(iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI;

(iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA;

(v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHII; or

(vi) brxP, brxC/pglY, pglZ and brxL

As used herein the term “isolated” refers to at least partially separated from the natural environment, physiological environment e.g., a microorganism e.g., bacteria.

As used herein “BREX system” (previously denoted “PYZA system”) or a “functional BREX system”, refers to a multi-gene system which comprises BRX and/or PGL genes which expression confers phage resistance.

According to specific embodiments, the BREX system is characterized by at least one of

(i) not being an abortive infection system;

(ii) not being a restriction modification system;

(iii) not preventing phage adsorption to a bacteria expressing same.

The BREX system may be characterized by one, two or all i.e.: (i); (ii); (iii); (i)+(ii); (i)+(iii); (ii)+(iii) and (i)+(ii)+(iii).

According to a specific embodiment the BREX system is characterized by (i)+(ii)+(iii).

As used herein “abortive infection (Abi) system” refers to a controlled cell death of an infected bacterial cell which takes place prior to the production of phage progeny, thus protecting the culture from phage propagation. Methods of analyzing Abi include, but are not limited to cell survival assays using high multiplicity of infection, one step growth assays and determination of phage DNA replication by e.g. DNA sequencing and southern blot analysis as further described hereinbelow.

As used herein “restriction modification system” refers to the recognition and cleavage of foreign DNA. Typically, a restriction modification system comprises a restriction enzyme having an activity of cleaving DNA and a modification enzyme capable of protecting host DNA from the cleavage by the restriction enzyme e.g. by methylating the host DNA. Analyzing restriction modification mode of action include, but is not limited to, evaluation of host specific methylation, presence of degraded foreign DNA and host cell death in the absence of the modification enzyme by methods described infra.

As used herein “adsorption” refers to the attachment to the host (e.g. bacteria) cell surface via plasma membrane proteins and glycoproteins. Methods of analyzing phage adsorption include, but are not limited to enumerating free phages in bacterial cultures infected with the phages immediately after phage addition and at early time points (e.g. 30 minutes) following phage addition as further described hereinbelow.

As used herein “phage resistance” refers to a phage infection resistance which can be a first or a second cycle resistance. The phage can be a lytic phage or a temperate (lysogenic) phage. According to a specific embodiment the BREX system confers phage resistance to a first cycle phage infection. According to yet other specific embodiments, BREX system confers resistance to lytic phages.

According to a specific embodiment, BREX system confers resistance to phage lysogeny.

As used herein, the term “lysogeny” refers to the incorporation of the phage genetic material inside the genome of the host (e.g. bacteria). Methods of analyzing phage lysogent are well known in the art and include, but not limited to, DNA sequencing and PCR analysis.

According to another specific embodiment, BREX system confers resistance to phage DNA replication.

According to specific embodiments. BREX system does not confer resistance to phages Φ105, rho10 and rho14.

As used herein, “phage resistance” refers to an increase of at least 10% in bacterial resistance towards a phage in comparison to bacteria of the same species under the same developmental stage (culture state) which does not express a BREX system, as may be manifested in e.g. bacterial viability, phage lysogeny and phage DNA replication. According to a specific embodiment, the increase is in at least 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.

Assays for testing phage resistance are well known in the art and mentioned hereinbelow.

According to specific embodiments, BREX system confers resistance to a plasmid. The plasmid may undergo integration into the bacterial genome or may be episomal.

According to a specific embodiment, the plasmid is episomal.

As used herein, “plasmid resistance” refers to an increase of at least 5% in bacterial resistance towards a plasmid in comparison to bacteria of the same species under the same developmental stage (culture state) which does not express a BREX system, as may be manifested in e.g. viability. According to a specific embodiment, the increase is in at least 10%, 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.

Assays for testing plasmid resistance are well known in the art and include, but not limited to, a transformation assay such as described in Itaya and Tsuge [Methods Enzymol (2011) 498:427-47].

As used herein, “expressing” or “expression” refers to gene expression at the RNA and/or protein level.

As used herein the “Phage growth Limitation” abbreviated as PGL refers to a cluster of genes which were previously described in Streptomyces coelicolor A3(2) (Chinenova T. A. et al, 1982; Sumby, P. & Smith, M. C. 2002, herein incorporated by reference in its entirety).

As used herein the “Bacteriophage Exclusion” abbreviated as BREX refers to a cluster of genes some of which were previously described in Streptomyces coelicolor A3(2) (Chinenova T. A. et al, 1982; Sumby, P. & Smith, M. C. 2002,).

According to specific embodiments, the BREX genes which compose the BREX system comprise brxC/pglY, pglZ pglW, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, which can be divided into six coherent subtypes comprising 4-8 genes each, in which the gene order and composition is conserved.

Thus, the BREX subtypes according to some embodiments of the present invention are selected from the group consisting of:

(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the functional BREX system does not comprise pglW.

(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI,

(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL (also may be referred to as Type 1).

(3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII (also may be referred to as Type 5),

(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI (also may be referred to as Type 6),

(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA (also may be referred to as Type 3),

(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI (also may be referred to as Type 2), or

(3vi) brxP, brxC/pglY, pglZ and brxL (also may be referred to as Type 4).

Thus, specific examples of BREX systems which can be used according to the present teachings include but are not limited to BREX system type 1, BREX system type 2, BREX system type 3. BREX system type 4, BREX system type 5 and BREX system type 6 (see FIG. 1A).

According to specific embodiments, BREX system type 1 (previously denoted PYZA system type 1a) comprises brxA, brxB, brxC/pglY, pglX, pglZ and brxL; BREX system type 5 (previously denoted PYZA system type 1b) comprises brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII; BREX system type 6 (previously denoted PYZA system type 1c) comprises brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI; BREX system type 3 (previously denoted PYZA system type 2) comprises brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA; BREX system type 2 (previously denoted PYZA system type 3) comprises pglW, pglX, brxC/pglY, pglZ, brxD and brxHI; and BREX system type 4 (previously denoted PYZA system type 4) comprises brxP, brxC/pglY, pglZ and brxL.

According to specific embodiments the BREX system is type 1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.

Two of the six genes found in type 1 BREX conserved cluster share homology with genes from the previously reported PGL system^(10,11): pglZ, coding for a protein with a predicted alkaline phosphatase domain, and pglX, coding for a protein with a putative methylase domain. The four additional genes include (i) a Ion-like protease-domain gene, denoted herein as brxL; (ii) brxA; (iii) brxB; and (iv) a ˜1200 amino acid protein with an ATP binding motif (GXXXXGK[T/S]), denoted herein as brxC. The preferential localization of this conserved gene cluster in the genomic vicinity of other defense genes suggests that it is a novel phage defense system.

The phage defense system originally described in Streptomyces coelicolor A3(2) as PGL is defined according to the present teachings as a type 2 BREX. While the PGL was described to be composed of four genes, pglW, pglX, pglY and pglZ, the present teaching suggest that 2 more genes, brxD and brxHI, are an integral part of the type 2 BREX system. In addition, pglW, an integral part of the previously described PGL, exists exclusively in type 2 BREX subtype.

The major phage resistance systems that were characterized to date, including the restriction-modification and CRISPR-Cas systems, encode mostly for proteins that interact with and manipulate DNA and RNA molecules. While the BREX system contains such proteins including methylases and helicases it also contains genes coding for proteins predicted to be involved in the manipulation of other proteins, such as the Ion-like protease, brxL, the predicted alkaline phosphatase, pglZ, and the serine/threonine kinase, brxW. Thus, according to specific embodiments, the defense mechanism employed by the BREX system takes place later in the infection where phage proteins are already produced and can be manipulated by pglZ and/or brxL.

According to other specific embodiments, BREX proteins target phage proteins co-injected with the phage DNA early in the infection cycle.

According to specific embodiments the BREX system acts before phage DNA replication.

According to specific embodiments, BREX proteins interact with other bacterial-encoded proteins to regulate BREX activity.

As used herein, the terms “pglY”, “brxC” and “brxC/pglY” refer to the polynucleotide and expression product e.g., polypeptide of the PGLY or BRXC gene. The polypeptide product of the PGLY and BRXC genes typically contains p-loop ATPase/ATP binding domain DUF2791 (pfam10923, SEQ ID NO: 6162) and a DUF499 domain. brxC/pglY together with pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, comprise a BREX system.

According to specific embodiments, brxC/pglY is selected from the group consisting of SEQ ID NO: 3155, 157-765 and 767-1175.

As used herein, the term “pglZ” refers to the polynucleotide and expression product e.g., polypeptide of the PGLZ gene. The polypeptide product of the PGLZ gene typically contains an alkaline phosphatase domain pfam08665. pglZ together with brxC/pglY and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, comprise a BREX system.

According to specific embodiments pglZ is selected from the group consisting of SEQ ID NO: 1176-1318, 1320-1856, 1858-2250, 6205 and 6204.

As used herein, the term “pglX” refers to the polynucleotide and expression product e.g., polypeptide of the PGLX gene. The polypeptide product of the PGLX gene typically contains an adenine-specific DNA methyltransferase domain pfam13659 (COG1002/COG0286). pglX together with at least brxC/pglY and pglZ and optionally at least one of pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system. pglX is a critical gene as the present inventors have shown that it presented high rates of irregularities in the BREX systems documented and a frame shift mutation in this gene in one of the type 1 BREX-containing Bacillus subtilis strains obtained was not active against any of the tested phages. In addition, Bacillus subtilis strains containing a type 1 BREX having a deletion of pglX were sensitive to all the phages tested.

According to specific embodiments pglX is selected from the group consisting of SEQ ID NO: 2251-3280 and 6186-6201.

According to specific embodiments, pglX is a methylase.

According to a specific embodiment, the pglX methylase and pglXI methylase are analogous in BREX systems types 1 and 3, respectively.

According to specific embodiments, the methylase of the BREX system methylates the bacterial DNA.

According to a specific embodiment, the methylase of the BREX system drives motif-specific (e.g. an adenine residue in TAGGAG motif) methylation on the genomic DNA of a bacteria expressing same. According to specific embodiments the methylation is non-polindromic. According to specific embodiments the BREX system methylase does not methylate a phage genome.

According to specific embodiments the methylation serves as part of the self/non-self recognition machinery of BREX.

According to a specific embodiment, type 4 BREX does not contain a methylase.

According to a specific embodiment the pglX methylase and brxP reductase are analogous in BREX systems types 1 and 4, respectively.

Methods of assessing DNA methylation and, more specifically, adenine-specific methylation are well known in the art and include e.g. the PacBio sequencing platform [Murray et al. Nucleic acids research (2012) 40: 11450-11462].

As used herein, the term “pglXI” refers to the polynucleotide and expression product e.g., polypeptide of the PGLXI gene. The polypeptide product of the PGLXI gene typically contains an adenine-specific DNA methylase COG0863/COG1743 (pfam 01555). pglXI together with at least brxC/pglY and pglZ and optionally at least one of pglX, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments pglXI is selected from the group consisting of SEQ ID NO: 3281-3296, 3298-3356 and 3358-3403.

As used herein, the term “brxP” (previously denoted “pglPA”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXP gene. The polypeptide product of the BRXP gene typically contains a phosphoadenosine phosphosulfate reductase domain COG0175 (pfam01507), and a pfam13182 domain. brxP together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxP is selected from the group consisting of SEQ ID NO: 3404-3440.

As used herein, the term “brxHI” (previously denoted “pglHI”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXHI gene. The polypeptide product of the BRXHI gene typically contains an Lhr-like a helicase domain COG1201. brxHI together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxHI is selected from the group consisting of SEQ ID NO: 3543-3642.

As used herein, the term “brxHII” (previously denoted “pglHII”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXHII gene. The polypeptide product of the BRXHII gene typically contains a DNA/RNA helicase domain COG0553. brxHII together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxHII is selected from the group consisting of SEQ ID NO: 3441-3460, 3462-3511, 3513-3542 and 6173-6185.

As used herein, the term “brxL” (previously denoted “pglL”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXL gene. The polypeptide product of the BRXL gene typically contains a ion-like protease domain COG4930. brxL together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxL is selected from the group consisting of SEQ ID NO: 3643-4412, 6165, 6166, 6169, 6170, 6202 and 6203.

As used herein, the term “brxD” (previously denoted “pglD”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXD gene. The polypeptide product of the BRXD gene typically contains an ATP binding domain DUF2791 (pfam10923). brxD together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxD is selected from the group of consisting SEQ ID NO: 4413-4488.

As used herein, the term “brxA” (previously denoted “pglA”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXA gene. The polypeptide product of the BRXA gene typically contains a DUF1819 (pfam08849) domain. The brxA protein displays significant structural homology to NusB spanning the RNA-binding interface, as well as part of the protein:protein interaction interface of NusB with NusE. In light of this similarity, according to specific embodiments brxA is an RNA binding protein. According to specific embodiments, brxA has a role in interfering with the phage infection cycle by disrupting anti-termination events essential for the phage cycle. brxA together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxB, brxF, brxE and pglW comprise a BREX system.

According to specific embodiments brxA is selected from the group consisting of SEQ ID NO: 4489-4621, 4623-5086, 5088-5415, 6167, 6168, 6171 and 6172.

As used herein, the term “brxB” (previously denoted “pglB”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXB gene. The polypeptide product of the BRXB gene typically contains a DUF1788 (pfam08747) domain. brxB together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP. brxHI, brxHII, brxL, brxD, brxA, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxB is selected from the group consisting of SEQ ID NO: 5416-5947 and 6206-6209.

As used herein, the term “brxF” (previously denoted “pglC”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXF gene. The polypeptide product of the BRXF gene typically contains an ATPase domain, brxF together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxE and pglW, comprise a BREX system.

According to specific embodiments brxF is selected from the group consisting of SEQ ID NO: 5948-5957, 5959-5988 and 5990-6028.

As used herein, the term “brxE” (previously denoted “pglE”) refers to the polynucleotide and expression product e.g., polypeptide of the BRXE gene. brxE together with at least brxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and pglW, comprise a BREX system.

According to specific embodiments brxE is selected from the group consisting of SEQ ID NO: 6029-6040.

As used herein, the term “pglW” refers to the polynucleotide and expression product e.g., polypeptide of the PGLW gene. The polypeptide product of the PGLW gene typically contains a serine/threonine kinase domain COG0515. pglW together with brxC/pglY, pglZ and pglX, and at least one of brxD and brxHI, and optionally at least one of pglXI, brxP, brxHII, brxL, brxA, brxB, brxF, and brxE, comprise a BREX system.

According to specific embodiments pglW is selected from the group consisting of SEQ ID NO: 6041-6138.

The terms “brxC/pglY”, “pglZ”, “pglX”, “pglXI”, “brxP”, “brxHI”, “brxHII”, “brxL”, “brxD”, “brxA”, “brxB”, “brxF”, “brxE”, and “pglW” also refers to functional brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW homologues which exhibit the desired activity (i.e., conferring phage resistance). Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polypeptide SEQ ID NO: 614-765, 767-1175, 1714-1856, 1858-2250, 6204, 2766-3280, 6186, 6188, 6190, 6192, 6194, 6196, 6198, 6200, 3343-3356, 3358-3403, 3422-3440, 3492-3511, 3513-3542, 6173, 6175, 6178, 6180, 6182, 6184, 3593-3642, 4028-4412, 6165, 6169, 6202, 4438-4488, 4953-5086, 5088-5415, 6167, 6171, 5570-5947, 6206, 6208, 5979-5988, 5990-6028, 6035-6040 and 6090-6138, respectively, or 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding same (as further described hereinbelow). The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.

Alternatively or additionally, homology can be based on shared motifs [e.g., the p-loop motif GXXXXGK(T/S) (DUF2791, SEQ ID NO: 6162) and DUF499 motifs present in pglY] combined with the conserved size of the gene in the different subtypes and the location of the gene in the gene cluster.

Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, MUSCLE, and HHpred.

As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

According to specific embodiments the polynucleotides of the present invention are part of a nucleic acid construct comprising the polynucleotide encoding the BREX system and at least one cis-acting regulatory element for directing expression of the nucleic acid sequence.

Teachings of the invention further contemplate that the polynucleotides are part of a nucleic acid construct system where the BREX genes are expressed from a plurality of constructs.

Thus, the present invention further provides for a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW.

The present invention further provides for a nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

Thus according to specific embodiments, the nucleic acid construct system comprises an individual nucleic acid construct for each BREX system pgl and/or brx gene.

According to other specific embodiments a single construct comprises a number of BREX system pgl and/or brx genes.

Cis acting regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of the nucleotide sequence only under certain conditions.

According to specific embodiments, the nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with some embodiments of the invention include for example the tetracycline-inducible promoter (Zabala M. et al., Cancer Res. 2004, 64(8): 2799-804) or pathogen-inducible promoters. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen.

According to specific embodiments the promoter is a bacterial nucleic acid (e.g., expression) construct.

A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence into mRNA. A promoter can have a transcription initiation region, which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter can also have a second domain called an operator, which can overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein can bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation can be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.

An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression can therefore be either positive or negative, thereby either enhancing or reducing transcription. Other examples of positive and negative regulatory elements are well known in the art. Various promoters that can be included in the protein expression system include, but are not limited to, a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambda promoter. Any suitable promoter can be used to carry out the present invention, including the native promoter or a heterologous promoter. Heterologous promoters can be constitutively active or inducible. A non-limiting example of a heterologous promoter is given in U.S. Pat. No. 6,242,194 to Kullen and Klaenhammer.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1987) Nature 198:1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al. (1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publication Nos. 36.776 and 121.775). The beta-lactamase (bla) promoter system (Weissmann, (1981) “The Cloning of Interferon and Other Mistakes,” in Interferon 3 (ed. 1. Gresser); bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128); the arabinose-inducible araB promoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. See also Balbas (2001) Mol. Biotech. 19:251-267, where E. coli expression systems are discussed.

In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or phage promoter can be joined with the operon sequences of another bacterial or phage promoter, creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433). For example, the tac (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol. Chem. 260:3539-3541) promoters are hybrid trp-lac promoters comprised of both trp promoter and lac operon sequences that are regulated by the lac repressor. The tac promoter has the additional feature of being an inducible regulatory sequence. Thus, for example, expression of a coding sequence operably linked to the tac promoter can be induced in a cell culture by adding isopropyl-1-thio-.beta.-D-galactoside (IPTG). Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The phage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of a phage promoter and an E. coli operator region (EPO Publication No. 267,851).

The nucleic acid construct can additionally contain a nucleotide sequence encoding the repressor (or inducer) for that promoter. For example, an inducible vector of the present invention can regulate transcription from the Lac operator (LacO) by expressing the nucleotide sequence encoding the LacI repressor protein. Other examples include the use of the lexA gene to regulate expression of pRecA, and the use of trpO to regulate ptrp. Alleles of such genes that increase the extent of repression (e.g., laclq) or that modify the manner of induction (e.g., lambda C1857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible) can be employed.

Various construct schemes can be utilized to express few genes from a single nucleic acid construct. For example, the genes can be co-transcribed as a polycistronic message from a single promoter sequence of the nucleic acid construct. To enable co-translation of all the genes from a single polycistronic message, the different polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule including the coding sequences of all the genes will be translated from both the capped 5′ end and the internal IRES sequence of the polycistronic RNA molecule to thereby produce the whole BREX system.

Alternatively, each two polynucleotide segments can be translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed with the nucleic acid construct. In this case, a chimeric polypeptide translated will be cleaved by the cell expressed protease to thereby generate the whole BREX system.

Still alternatively, the nucleic acid construct of some embodiments of the invention can include at least two promoter sequences each being for separately expressing a specific pgl or brx. These at least two promoters which can be identical or distinct can be constitutive, tissue specific or regulatable (e.g. inducible) promoters functional in one or more cell types.

The nucleic acid construct (also referred to herein as an “expression vector” or a “vector”) of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, typical vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.

When secretion of the polypeptides is desired the polynucleotides of the invention can be expressed as fusion polypeptides comprising the nucleic acid sequence encoding the PGL or BRX gene ligated in frame to a nucleic acid sequence encoding a signal peptide that provides for secretion.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb et al. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212). Other prokaryotic signals include, for example, the signal sequence from penicillinase, Ipp, or heat stable enterotoxin II leaders.

According to a specific embodiment, the nucleic acid construct comprises a plurality of cloning sites for ligating a nucleic acid sequence of the invention such that it is under transcriptional regulation of the regulatory regions.

Selectable marker genes that ensure maintenance of the vector in the cell can also be included in the expression vector. Preferred selectable markers include those which confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers can also allow a cell to grow on minimal medium, or in the presence of toxic metabolite and can include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed polypeptide.

Where appropriate, the polynucleotides may be optimized for increased expression in the transformed organism. For example, the polynucleotides can be synthesized using preferred codons for improved expression.

Various methods known within the art can be used to introduce the expression vector of some embodiments of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, natural or induced transformation, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

Exemplary methods of introducing expression vectors into bacterial cells include for example conventional transformation or transfection techniques, or by phage-mediated infection. As used herein, the terms “transformation”, “transduction”, “conjugation”, and “protoplast fusion” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a cell, such as calcium chloride co-precipitation.

Introduction of nucleic acids by phage infection offers several advantages over other methods such as transformation, since higher transfection efficiency can be obtained due to the infectious nature of phages. These methods are especially useful for rendering bacteria more sensitive to phage attack for antibiotics purposes as further described hereinbelow.

It will be appreciated the BREX polypeptides can be introduced directly into the cell (e.g., bacterial cell) and not via recombinant expression to confer resistance. The term “polypeptide” as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.

The polypeptides of the present invention may be synthesized by any techniques known to those skilled in the art of peptide synthesis, for example but not limited to recombinant DNA techniques or solid phase peptide synthesis.

Thus, regardless of the method of introduction, the present teachings provide for an isolated cell (e.g., bacterial cell) which comprises a heterologous BREX system, as described herein.

According to specific embodiments, the isolated cell is transformed or transfected with the above-mentioned nucleic acid construct or nucleic acid construct system.

According to an aspect of the present invention, there is provided an isolated cell genetically modified to express a BREX system selected from the group consisting of

(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHI, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW,

(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI,

(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL.

(3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII.

(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI,

(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA,

(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or

(3vi) brxP, brxC/pglY, pglZ and brxL.

According to another aspect of the present invention there is provided an isolated cell genetically modified to express a BREX system polypeptide selected from the group consisting of pglX, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.

According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of a COG0515 domain, said polypeptide conferring resistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of a pfam13659 domain, said polypeptide conferring resistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of DUF2791 and DUF499 domains, said polypeptide conferring resistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cell genetically modified to express a BREX system polypeptide comprising an amino acid sequence of a pfam08665 domain, said polypeptide conferring resistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cell genetically modified to express a pglW polypeptide with the proviso that said pglW polypeptide is not SEQ ID NO: 6110.

According to specific embodiments there is provided an isolated cell genetically modified to express a pglX polypeptide with the proviso that said pglX polypeptide is not SEQ ID NO: 2949.

According to specific embodiments there is provided an isolated cell genetically modified to express a brxC/pglY polypeptide with the proviso that said brxC/pglY polypeptide is not SEQ ID NO: 802.

According to specific embodiments there is provided an isolated cell genetically modified to express a pglZ polypeptide with the proviso that said pglZ polypeptide is not SEQ ID NO: 1890.

According to specific embodiment the isolated cell (e.g., bacterial cell) does not express a BREX system endogenously.

The term “endogenous” as used herein, refers to the expression of the native gene in its natural location and expression level in the genome of an organism.

The expression of the polynucleotide can be episomal or integrated into the chromosome of the cell.

According to specific embodiments the isolated cell is resistant to a first cycle phage infection.

According to specific embodiments the isolated cell is resistant to lytic phage.

According to specific embodiments the isolated cell is resistant to temperate (also referred as lysogenic) phage.

According to a specific embodiment the isolated cell is resistant to phage lysogeny.

According to another specific embodiment the isolated cell is resistant to phage DNA replication.

According to specific embodiments the isolated cell is a microbial cell such as a bacterial cell.

As used herein, the term “bacteria” refers to all prokaryotes and includes both bacteria and archaea.

Indeed, it is intended that any bacterial species (e.g., which does not express a PYZA system) will find use in the present invention. Thus, the bacteria may be for example gram-positive or gram-negative bacteria.

The phrase “Gram-positive bacteria” as used herein refers to bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure. Representative Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens. Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae. Corvnebacteriwnum jeikeium, Enterococcus faecalis, Enterococcus faecium, Ervsipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex. Mycobacterium chelonae, Mycobacterium fortuitum. Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis. Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus colmii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus. Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis. Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans. Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” as used herein refers to bacteria characterized by the presence of a double membrane surrounding each bacterial cell. Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xvlosoxidans, Bacteroides. Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamndia psittaci. Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides. Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis.

According to specific embodiments the bacteria is a species selected from the group consisting of Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, Bifidobacterium, Brevibacterium, Propionibacterium. Lactococcus, Lactobacillus, Pediococcus, Leuconostoc. Oenococcus, and Propionibacterium species.

Additionally, or alternatively the bacteria may be useful in the manufacture of dairy and fermentation processing such as, but not limited to, milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk.

According to specific embodiments the bacteria is a lactic bacteria. As used herein the term “lactic acid bacteria” refers to Gram positive, microaerophillic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid.

According to specific embodiments the bacteria is a species selected from the group of the industrially most useful lactic acid bacteria consisting of Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species, Pediococcus species and Bifidobacterium species and Propionibacterium species.

As used herein, the term “phage” or “bacteriophage” refers to a virus that selectively infects one or more bacterial species. Many phages are specific to a particular genus or species or strain of bacteria.

According to specific embodiments, the phage is virulent to the bacteria.

According to some embodiments, the phage is a lytic phage.

According to other embodiments, the phage is temperate (also referred to as lysogenic).

A lytic phage is one that follows the lytic pathway through completion of the lytic cycle, rather than entering the lysogenic pathway. A lytic phage undergoes viral replication leading to lysis of the cell membrane, destruction of the cell, and release of progeny phage particles capable of infecting other cells.

A temperate phage is one capable of entering the lysogenic pathway, in which the phage becomes a dormant, passive part of the cell's genome through prior to completion of its lytic cycle.

Exemplary phages which fall under the scope of the invention include, but are not limited to, phages that belong to any of the following virus families: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae.

According to specific embodiments the phage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T, and SPO2.

According to other specific embodiments the phage is not Φ105, rho10 and rho14.

According to specific embodiments, the lytic phage is SPO1 and/or SP82G.

According to specific embodiments, phage that infect bacteria that are pathogenic to plants and/or animals (including humans) find particular use.

According to specific embodiments, the resistance of a cell against a phage is improved as compared to a cell of the same species which was not treated according to the present teachings (i.e., with a BREX system).

The lysogenic activity of a phage can be assessed in multiple ways, including but not limited to PCR and DNA sequencing.

The DNA replication activity of a phage can be assessed in multiple ways, including but not limited to DNA sequencing and southern blot analysis.

The lytic activity of a phage can be assessed in multiple ways, including but not limited to optical density, plaque assay, and living dye indicators.

The lytic activity of a phage can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. This method involves introduction of phage into a fluid bacterial culture medium. After a period of incubation, the phage lyses the bacteria in the broth culture resulting in a clearing of the fluid medium resulting in decrease in optical density.

Another method, known as the plaque assay, introduces phage into a few milliliters of soft agar along with some bacterial host cells. This soft agar mixture is laid over a hard agar base (seeded-agar overlay). The phage adsorb onto the host bacterial cells, infect and lyse the cells, and then begin the process anew with other bacterial cells in the vicinity. After 6-24 hours, zones of clearing on the plate known as plaques, are observable within the lawn of bacterial growth on the plate. Each plaque represents a single phage particle in the original sample.

Yet another method is the one-step phage growth curve which allows determining the production of progeny virions by cells as a function of time after infection. The assay is based on the fact that cells in the culture are infected simultaneously with a low number of phages so that no cell can be infected with more than one phage. At various time intervals, samples are removed for a plaque assay allowing quantitative determination of the number of phages present in the medium.

Other methods use for example redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a dye (e.g., tetrazolium dye) and produces a color change that can be measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color.

Thus, the polynucleotides, polypeptides and nucleic acid constructs of the present invention can be used in conferring phage resistance.

As used herein, “confers phage resistance” refers to an increase of at least 10% in bacterial resistance towards a phage, as may be manifested in viability. According to a specific embodiment, the increase is in at least 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.

For the same culture conditions the bacterial susceptibility towards a phage of the present invention is generally expressed in comparison to the wild-type bacteria. As used herein, the phrase “increased resistance towards a phage” means that the level of phage infection and/or multiplication in the bacteria does not cause a deleterious effect to the bacteria e.g., growth arrest or death.

In some embodiments, the bacteria have about 100-100.000 times lower efficiency of plaquing ([EOP]=10-2), about 1000 times lower EOP (EOP=10⁼³), 10,000 times lower EOP (EOP=10-4), or 100,000 times lower EOP (EOP=10-5). In some embodiments, the level of phage multiplication in a culture is measured after about 6-14 hours incubation of the culture, e.g., after about 12 hours, after about 9 hours, after about 8 hours after about 7 hours, or after about 6 hours.

Thus, according to specific embodiments there is provided a method of protecting bacteria from phage attack, the method comprising introducing into or expressing in the bacteria a BREX system, thereby protecting the bacteria from phage attack.

According to specific embodiments the bacteria does not express a BREX system endogenously.

Various modalities may be used to introduce or express the BREX system in the bacteria.

Thus, according to specific embodiments, the method is effected by expressing in the bacteria, the isolated polynucleotides, nucleic acid construct or construct system or alternatively introducing the BREX polypeptides as described herein to confer protection.

According to another embodiment the BREX system is introduced into the bacteria via a transmissible genetic element in a process of bacterial conjugation.

As used herein, the phrase “bacterial conjugation” refers to a direct transfer of genetic material between bacterial cells by cell-to-cell contact or by bridge-like connection between the cells. During conjugation the donor bacteria provides a transmissible genetic element, typically a plasmid or a transposon. The transfer of the transmissible genetic element tale advantage of the complementary nature of double stranded DNA. Thus, one strand of the transmissible genetic element is transferred and the other remains in the original bacteria. Both strands have the complementary stranded added so that each bacteria ends up with a complete transmissible element.

According to a specific embodiment, there is provided a method of protecting first bacteria from phage attack, the method comprising contacting the first bacteria with second bacteria which expresses on a transmissible genetic element a BREX system, wherein the first bacteria and the second bacteria are non identical; thereby protecting the bacteria from phage attack.

As used herein, the term “contacting” refers to the step of incubation of the bacterial cell (e.g., first bacteria) with a substance or cell (e.g., second bacteria) such that the substance or a substance contained in the cell affects phage resistance of the bacterial cell.

According to specific embodiments the first bacteria does not express a BREX system endogenously.

As used herein the phrase “transmissible genetic element” refers to a nucleic acid sequence that can be transferred naturally from one bacteria to another.

According to specific embodiments the transmissible genetic element comprises a conjugative genetic element or a conjugative plasmid or mobilizable genetic element.

As used herein, a “conjugative plasmid” refers to a plasmid that is transferred from one bacterial cell to another during conjugation.

As used herein, the term “mobilizable element” refers to a transposon, which is a DNA sequence that can change its position within the genome.

According to a specific embodiment, the first bacteria is the industrially valuable bacteria such as those used for fermentation as described above.

Thus, following the above teachings there is provided an isolated bacteria comprising a nucleic acid sequence encoding a BREX system and a transmissible genetic element expressing the BREX system, wherein the isolated bacteria does not endogenously express the BREX system and wherein the BREX system comprises brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprises brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

Cultures, and starter cultures, in particular are used extensively in the food industry in the manufacture of fermented products including milk products (e.g., yogurt, buttermilk, and cheese), meat products, bakery products, wine, and vegetable products. The preparation of cultures is labor intensive, occupying much space and equipment, and there is a considerable risk of contamination with spoilage bacteria and/or phages during the propagation steps. The failure of bacterial cultures due to phage infection and multiplication is a major problem with the industrial use of bacterial cultures. There are many different types of phages and new strains continue to emerge. Indeed, despite advances in culture development, there is a continuing need to improve cultures for use in industry.

Thus, according to an aspect of the present invention, there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product the isolated BREX system polynucleotide, the BREX system construct, the isolated cell or the isolated bacteria of the present invention, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.

Thus, following the above teachings there is provided a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising the isolated BREX system polynucleotide, the BREX system construct, the isolated cell or the isolated bacteria of the present invention.

According to another aspect of the present invention, there is provided a method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to the food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product a bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.

According to specific embodiments the food or feed is a dairy product.

The preparation of starter cultures of such bacteria, and methods of fermenting substrates, particularly food substrates such as milk, can be carried out in accordance with known techniques, including but not limited to those described in Mayra-Makinen and Bigret (1993) Lactic Acid Bacteria; Salminen and vonWright eds. Marcel Dekker, Inc. New York. 65-96; Sandine (1996) Dairy Starter Cultures Cogan and Accolas eds. VCH Publishers, New York. 191-206; Gilliland (1985) Bacterial Starter Cultures for Food. CRC Press, Boca Raton. Fla.

The term “fermenting” refers to the energy-yielding, metabolic breakdown of organic compounds by microorganisms that generally proceeds under anaerobic conditions and with the evolution of gas.

Products produced by fermentation which have been known to experience phage infection, and the corresponding infected fermentation bacteria, include cheddar and cottage cheese (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris), yogurt (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus). Swiss cheese (S. thermophilus, Lactobacillus lactis, Lactobacillus helveticus), blue cheese (Leuconostoc cremoris), Italian cheese (L. bulgaricus, S. thermophilus), viili (Lactococcus lactis subsp. cremoris. Lactococcus lactis subsp. lactis biovar diacetylactis, Leuconostoc cremoris), yakult (Lactobacillus casei), casein (Lactococcus lactis subsp. cremoris), natto (Bacillus subtilis var. natto), wine (Leuconostoc oenos), sake (Leuconostoc mesenteroides), polymyxin (Bacillus polymyxa), colistin (Bacillus colistrium), bacitracin (Bacillus licheniformis), L-glutamic acid (Brevibacterium lactofermentum, Microbacterium ammoniaphilum), and acetone and butanol (Clostridium acetobutylicum. Clostridium saccharoperbutvlacetonicum).

The present inventors have uncovered that transformation of a Bacillus subtilis strain with a non-complete type 1 BREX (i.e. not expressing pglX) does not confer phage resistance. In addition it was also discovered that a frame shift mutation in a BREX gene (i.e., pglX) in one of the Bacillus subtilis strains transformed with type 1 BREX resulted in aberrant BREX system that was not active against any of the tested phages, indicating that down regulation of a BREX gene can render a bacteria resistant to phage infection. These results suggest the use of anti BREX agents as a method to induce phage sensitivity.

As used herein, “inducing phage sensitivity” refers to an increase of at least 10% in bacterial susceptibility towards a phage, as may be manifested in growth arrest or death. According to a specific embodiment, the increase is in at least 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or more than 100%.

For the same culture conditions, the bacterial susceptibility towards a phage of the present invention is generally expressed in comparison to the wild-type bacteria. As used herein, the phrase “increased susceptibility towards a phage” means that the level of phage infection and/or multiplication in the bacteria cause a deleterious effect to the bacteria e.g., growth arrest or death.

Thus, according to further aspect of the present invention, there is provided a method of inducing phage sensitivity in a bacterial cell, the method comprising contacting a bacterial cell which expresses a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglX, brxP, brxHI, brxHI, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducing sensitivity of the bacterial cell to phage infection.

As used herein the phrase “anti BREX system agent” is an agent capable of specifically inhibiting or “silencing” the expression of a target BREX gene or alternatively specifically impairs the functionality of the target BREX protein. According to specific embodiments the anti BREX system agent is directed against pglX. For example, the anti-BREX system may interfere with pglX expression (as described hereinbelow) or in its DNA methyltransferase function by the use of common inhibitors of such an enzyme e.g., 5-Azacytidine. Decitabine Zebularine, RG108, Hydralazine hydrochloride, and Psammaplin A.

According to other specific embodiments the anti BREX system agent is directed against brxC/pglY or pglZ.

Down regulation of BREX system can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents), or on the protein level using e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide and the like.

According to specific embodiments the anti BREX system agent is selected from the group consisting of a nucleic acid suitable for silencing expression, aptamers, small molecules and inhibitory peptides.

As used herein the phrase “nucleic acid suitable for silencing expression” refers to regulatory mechanisms mediated by nucleic acid molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. Numerous methods are known in the art for gene silencing in prokaryotes. Examples include but are not limited to U.S. Patent Application 20040053289 which teaches the use of si hybrids to down-regulate prokaryotic genes, and

U.S. Patent Application PCT/US09/69258 which teaches the use of CRISPR to downregulate prokaryotic genes. Alternatively the inhibition can be carried out at the protein level which interferes with protein activity, such as by the use of aptamers. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).

As used herein an “aptamer” refers to double stranded DNA or single stranded RNA molecule that binds to specific molecular target, such as a protein.

Alternatively or additionally, small molecule or peptides can be used which interfere with the BREX protein function (e.g., catalytic or interaction).

Specifically, contacting is effected such that the positioning of the anti BREX system agent is in direct or indirect contact with the bacterial cell. Thus, the present invention contemplates both applying the anti BREX system agents of the present invention to a desirable surface and/or directly to the bacterial cells.

According to another embodiment the surface is comprised in a biological tissue, such as for example, mammalian tissues e.g. the skin.

It will be appreciated that the bacteria may be comprised inside a particular organism, (e.g. intracellularly or extracellularly) for example inside a mammalian body or inside a plant. In this case, the contacting may be effected by administering the anti BREX agents per se or by transfecting the cells of the organism with the anti BREX agents of the present invention.

Thus, according to a specific embodiment contacting with an anti BREX system agent is effected in-vivo.

According to another specific embodiment contacting with an anti BREX system agent is effected ex-vivo.

According to another specific embodiment contacting with an anti BREX system agent is effected in-vitro.

According to specific embodiments, there is provided an isolated bacteria generated by contacting bacteria with anti BREX system agent in-vitro or ex-vivo.

According to some embodiments, a BREX system or an anti-BREX system agent is provided in a formulation suitable for cell penetration that enhances intracellular delivery of BREX system.

Any suitable penetrating agent for enhancing penetration of BREX system or anti BREX system agent to cell (e.g., bacteria) may be used, as known by those of skill in the art. Examples include but are not limited to:

Phages—Phages offer several advantages including lateral infection, higher efficiency of transformation, and targeting to, and propagation in, specific bacteria.

Cell-Penetrating Peptides (CPPs)—CPPs, for example TAT (transcription activator from HIV-1) are short peptides (≦40 amino acids), with the ability to gain access to the interior of almost any cell. They are highly cationic and usually rich in arginine and lysine amino acids. They have the exceptional property of carrying into the cells a wide variety of covalently and noncovalently conjugated cargoes such as proteins, oligonucleotides, and even 200 nm liposomes. Protocols for producing CPPs-cargos conjugates and for infecting cells with such conjugates can be found, for example L. Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91:664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626-1633].

The expression level and/or activity level of the BREX system expressed in the cells of some embodiments of the invention can be determined using methods known in the arts, e.g. but not limited to selectable marker gene, Northern blot analysis, PCR analysis, DNA sequencing, RNA sequencing, Western blot analysis, and Immunohistochemistry.

According to another aspect of the present invention, there is provided a method of treating a microbial infection in a subject in need thereof, the method comprising contacting the bacteria with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby treating the infection.

As used herein, the term “treating” refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection.

As used herein, the phrase “subject in need thereof” includes mammals, preferably human beings at any age which suffer from pathogen infection.

The anti BREX system agent may be used alone or together with additional antimicrobial agents (e.g. phage therapy, antibiotic and/or additional anti microbial peptides).

According to specific embodiments the methods of the present invention further comprise administering to the subject a phage therapy.

According to other specific embodiments the methods of the present invention further comprise administering to the subject an antibiotic.

Exemplary antibiotics include, but are not limited to aminoglycoside antibiotics, cephalosporins, quinolone antibiotics, macrolide antibiotics, penicillins, sulfonamides, tetracyclines and carbapenems. It will be appreciated that since the polypeptides of embodiments of this invention enhance the antibacterial effect of the antibiotic, doses of the antibiotic may be lower (e.g. 20% lower, 30% lower, 40% lower, 50% lower, 60% lower, 70% lower, 80% lower or even 90% lower than those currently in use.

The BREX system or the anti-BREX system agent of some embodiments of the invention can be administered to a starter culture, a fermentation vat or an organism per se, or in a composition where it is mixed with suitable carriers or excipients.

According to an aspect of the present invention there is provided a phage defense composition, comprising as an active ingredient a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHII, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; and an acceptable carrier or diluent.

According to another aspect of the present invention there is provided an anti-microbial composition comprising as active ingredient an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and an acceptable carrier or diluent.

As used herein, the phrase “anti-microbial activity”, refers to an ability to suppress, control, inhibit or kill a bacteria. Thus, for example the anti-microbial activity may comprise bactericidal or bacteriostatic activity, or both.

According to specific embodiments the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

The phrase “pharmaceutical composition” as used herein refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

As used herein the term “active ingredient” refers to any one of BREX system polypeptide or polynucleotide, anti-BREX system agent capable of down regulating a BREX gene or cells generated according to the present teachings, accountable for the biological effect.

Techniques for formulation and administration of drugs may be found in the latest edition of “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., which is herein fully incorporated by reference and are further described herein below.

It will be appreciated that the polypeptides, polynucleotides, or other agents of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.

Exemplary additional agents include phage therapy, and antibiotics (e.g. rifampicin, chloramphenicol and spectinomycin).

According to specific embodiment the anti-microbial composition further comprises a phage.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

The preparation of the present invention may also be formulated as a topical composition, such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste, a gel and a patch.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease (e.g., bacterial infection) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1].

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

According to another aspect there is provided an article of manufacture or a kit identified for killing a bacteria comprising a packaging material packaging an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and a phage.

According to specific embodiments the anti BREX system agent and the phage are packaged in separate containers.

According to yet other specific embodiments the anti BREX system agent and the phage are in c-formulation.

According to further aspect of the present invention there is provided a method of screening for identifying phage useful for infecting a bacteria, the method comprising:

(a) contacting a phage with a bacteria expressing BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREX system does not comprise pglW or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI;

(b) monitoring phage sensitivity of the bacteria, wherein an increase in phage sensitivity of the bacteria in the presence of the phage compared to phage sensitivity in the absence of the phage is indicative of a phage useful for infecting the bacteria.

The method comprising further isolating the phage characterizing it in terms of sequencing and compatibility with phages species and the ability to infect different bacterial species.

Tables 2-8 and 10-16 below demonstrate the six types of BREX system in a diverse array of bacteria and archaea genomes.

TABLE 2 BREX type 1 systems Genomic Genomic Start End Methylase Replicon Organism Point Point BrxA* BrxB* BrxC* PglX* PglZ* BrxL* state Type Acidiphilium 497519 512043 YP_004282676.1/ YP_004282675.1/ YP_004282674.1/ YP_004282672.1/ YP_004282670.1/ YP_004282669.1/ Split chromosome multivorum AIU301 4953 5570 617 2766 1716 4300 methylase. uid63345 YP_004282673.1/ 2767 Acidithiobacillus 2361379 2377748 YP_004784791.1/ YP_004784792.1/ YP_004784793.1/ YP_004784794.1/ YP_004784799.1/ YP_004784800.1/ Split chromosome ferrivorans SS3 uid67387 4954 5571 618 2768 1717 4301 methylase. YP_004784796.1/ 2769 Acinetobacter baumannii 53842 68427 YP_001708756.1/ YP_001708755.1/ YP_001708754.1/ YP_001708753.1/ YP_001708751.1/ YP_001708750.1/ plasmid AYE uid61637 4955 5572 619 2770 1718 4302 Anaeromyxobacter 3688396 3701032 YP_466441.1/ YP_466440.1/ YP_466439.1/ YP_466438.1/ YP_466437.1/ YP_466436.1/ chromosome dehalogenans 2CP C 4957 5574 621 2772 1720 4303 uid58135 Aromatoleum 3091305 3115716 YP_159986.1/ YP_159987.1/ YP_159988.1/ YP_159991.1/ YP_160004.1/ YP_160005.1/ chromosome aromaticum EbN1 4958 5575 622 2773 1721 4304 uid58231 Burkholderia CCGE1001 3119206 3136704 YP_004229224.1/ YP_004229223.1/ YP_004229222.1/ YP_004229221.1/ YP_004229217.1/ YP_004229216.1/ chromosome uid42975 4963 5580 627 2777 1726 4309 Burkholderia gladioli 1574842 1588056 YP_004360051.1/ YP_004360052.1/ YP_004360053.1/ YP_004360054.1/ YP_004360055.1/ YP_004360056.1/ chromosome BSR3 uid66301 4964 5581 628 2778 1727 4310 Burkholderia 2201162 2216554 YP_001119856.1/ YP_001119855.1/ YP_001119854.1/ Missing YP_001119851.1/ YP_001119850.1/ chromosome vietnamiensis G4 4965 5582 629 1728 4311 uid58075 Calditerrivibrio 2095970 2108863 YP_004052086.1/ YP_004052085.1/ YP_004052084.1/ YP_004052081.1/ YP_004052080.1/ YP_004052079.1/ Split chromosome nitroreducens DSM 4966 5583 630 2779 1729 4312 methylase. 19672 uid60821 YP_004052083.1/ 2780 Carboxydothermus 2348825 2361903 YP_361448.1/ YP_361447.1/ YP_361446.1/ YP_361445.1/ YP_361444.1/ YP_361443.1/ chromosome hydrogenoformans Z 4967 5584 631 2781 1730 4313 2901 uid57821 Chlorobium 1811542 1826075 YP_001960082.1/ YP_001960081.1/ YP_001960080.1/ YP_001960077.1/ YP_001960076.1/ YP_001960075.1/ chromosome phaeobacteroides BS1 4968 5585 632 2782 1731 4314 uid58131 Clostridium ljungdahlii 3566681 3580351 YP_003781421.1/ YP_003781420.1/ YP_003781419.1/ YP_003781418.1/ YP_003781417.1/ YP_003781415.1/ chromosome DSM 13528 uid50583 4971 5588 635 2786 1734 4317 Clostridium 3014510 3029581 YP_003822961.1/ YP_003822960.1/ YP_003822959.1/ YP_003822957.1/ YP_003822954.1/ YP_003822953.1/ chromosome saccharolyticum WM1 4972 5589 636 2787 1735 4318 uid51419 Clostridium 3993474 4020337 YP_003823839.1/ YP_003823838.1/ YP_003823837.1/ YP_003823836.1/ YP_003823832.1/ YP_003823831.1/ chromosome saccharolyticum WM1 4973 5590 637 2788 1736 4319 uid51419 Clostridium sticklandii 1172754 1188586 YP_003936059.1/ YP_003936060.1/ YP_003936061.1/ YP_003936062.1/ YP_003936065.1/ YP_003936066.1/ chromosome DSM 519 uid59585 4974 5591 638 2790 1737 4320 Clostridium SY8519 1975332 1994112 YP_004708906.1/ YP_004708907.1/ YP_004708908.1/ YP_004708910.1/ YP_004708913.1/ YP_004708914.1/ chromosome uid68705 4975 5592 639 2791 1738 4321 Cupriavidus necator N 1 3467559 3485436 YP_004687115.1/ YP_004687114.1/ YP_004687113.1/ YP_004687110.1/ YP_004687108.1/ YP_004687107.1/ chromosome uid68689 4976 5593 640 2792 1739 4322 Cyanothece PCC 8802 2210340 2225482 Missing YP_003137927.1/ YP_003137926.1/ YP_003137924.1/ YP_003137920.1/ YP_003137919.1/ One chromosome uid59143 5594 641 2794 1740 4323 complete YP_003137923.1/ methylase 2795 and one truncated. Dehalococcoides VS 244220 257903 YP_003329752.1/ YP_003329751.1/ YP_003329750.1/ YP_003329749.1/ YP_003329748.1/ chromosome uid42393 4977 642 2795 1741 4324 Dehalogenimonas 1589660 1603624 YP_003759227.1/ YP_003759226.1/ YP_003759225.1/ YP_003759224.1/ YP_003759223.1/ YP_003759222.1/ chromosome lykanthroporepellens BL 4978 5595 643 2796 1742 4325 DC 9 uid48131 Desulfitobacterium 792814 806836 YP_516908.1/ YP_516909.1/ YP_516910.1/ YP_516912.1/ YP_516913.1/ chromosome hafniense Y51 uid58605 4979 644 2797 1743 4326 Desulfomicrobium 1457033 1471353 YP_003157843.1/ YP_003157842.1/ YP_003157841.1/ YP_003157840.1/ YP_003157838.1/ YP_003157837.1/ chromosome baculatum DSM 4028 4981 5597 646 2799 1745 4328 uid59217 Desulfovibrio 4090474 4104148 YP_002954994.1/ YP_002954993.1/ YP_002954992.1/ YP_002954991.1/ YP_002954989.1/ YP_002954988.1/ chromosome magneticus RS 1 4983 5599 648 2801 1747 4330 uid59309 Desulfovibrio vulgaris 2096179 2110453 Missing YP_011241.1/ YP_011240.1/ YP_011237.1/ YP_011235.1/ YP_011234.1/ chromosome Hildenborough uid57645 5600 649 2802 1748 4331 Desulfurivibrio 2512936 2529324 YP_003691455.1/ YP_003691454.1/ YP_003691453.1/ YP_003691451.1/ YP_003691448.1/ YP_003691447.1/ chromosome alkaliphilus AHT2 4985 5602 651 2804 1750 4333 uid49487 Erwinia pyrifoliae Ep1 3075240 3088692 YP_002649786.1/ YP_002649787.1/ YP_002649788.1/ YP_002649789.1/ YP_002649790.1/ YP_002649791.1/ chromosome 96 uid40659 4989 5606 655 2809 1754 4337 Erythrobacter litoralis 2963192 2977694 YP_459832.1/ YP_459834.1/ YP_459835.1/ YP_459838.1/ YP_459839.1/ YP_459840.1/ chromosome HTCC2594 uid58299 4990 5607 656 2810 1755 4338 Escherichia coli HS 335287 354281 YP_001457107.1/ YP_001457108.1/ YP_001457109.1/ YP_001457110.1/ YP_001457111.1/ YP_001457112.1/ chromosome uid58393 4993 5610 659 2814 1758 4341 Escherichia coli O111 H 5250834 5264277 YP_003237428.1/ YP_003237427.1/ YP_003237426.1/ YP_003237425.1/ YP_003237424.1/ YP_003237423.1/ chromosome 11128 uid41023 4994 5611 660 2815 1759 4342 Escherichia fergusonii 13818 28889 YP_002394569.1/ YP_002394570.1/ YP_002394571.1/ YP_002394573.1/ YP_002394574.1/ YP_002394575.1/ plasmid ATCC 35469 uid59375 4995 5612 661 2816 1760 4343 Exiguobacterium 325862 338926 YP_001812815.1/ YP_001812816.1/ YP_001812817.1/ YP_001812818.1/ YP_001812819.1/ YP_001812820.1/ chromosome sibiricum 255 15 4996 5613 662 2817 1761 4344 uid58053 Gallionella 927586 949287 YP_003846678.1/ YP_003846679.1/ YP_003846680.1/ YP_003846683.1/ YP_003846691.1/ YP_003846692.1/ chromosome capsiferriformans ES 2 4998 5615 664 2819 1763 4346 uid51505 Gallionella 1651741 1666042 YP_003847303.1/ YP_003847304.1/ YP_003847305.1/ YP_003847308.1/ YP_003847309.1/ YP_003847310.1/ chromosome capsiferriformans ES 2 4999 5616 665 2820 1764 4347 uid51505 Geobacillus WCH70 1353239 1364077 YP_002949411.1/ YP_002949412.1/ YP_002949413.1/ YP_002949414.1/ YP_002949415.1/ Missing chromosome uid59045 5000 5617 666 2821 1765 Geobacter sulfurreducens 2316576 2330995 NP_953159.2/ NP_953158.1/ NP_953157.1/ NP_953156.1/ NP_953155.1/ NP_953154.1/ chromosome PCA uid57743 5001 5618 667 2822 1766 4348 Haliscomenobacter 16081 32343 YP_004451379.1/ YP_004451378.1/ YP_004451377.1/ YP_004451376.1/ YP_004451373.1/ YP_004451372.1/ plasmid hydrossis DSM 1100 5002 5619 668 2823 1767 4349 uid66777 Lactobacillus casei 2008786 2025228 YP_003789119.1/ YP_003789118.1/ YP_003789117.1/ YP_003789114.1/ YP_003789113.1/ YP_003789112.1/ One chromosome Zhang uid50673 5007 5624 673 2828 1772 4351 complete YP_003789116.1/ methylase 2829 and one truncated Lactobacillus johnsonii 939272 953168 YP_003293166.1/ YP_003293165.1/ YP_003293164.1/ YP_003293163.1/ YP_003293159.1/ Missing chromosome FI9785 uid41735 5010 5627 676 2832 1775 Lactobacillus reuteri 1310070 1322351 YP_004649803.1/ YP_004649804.1/ YP_004649805.1/ YP_004649806.1/ YP_004649809.1/ Missing chromosome SD2112 uid55357 5011 5628 677 2833 1776 Lactobacillus rhamnosus 2154005 2170387 YP_003171846.1/ YP_003171845.1/ YP_003171844.1/ YP_003171841.1/ YP_003171840.1/ YP_003171839.1/ One chromosome GG uid59313 5013 5630 679 2836 1778 4353 complete YP_003171843.1/ methylase 2837 and one truncated. Leuconostoc kimchii 34362 50836 YP_003620562.1/ YP_003620561.1/ YP_003620560.1/ YP_003620557.1/ YP_003620556.1/ YP_003620555.1/ One chromosome IMSNU 11154 uid48589 5014 5631 680 2838 1779 4354 complete YP_003620559.1/ methylase 2839 and one truncated. Magnetospirillum 2215526 2231932 YP_421401.1/ YP_421402.1/ YP_421403.1/ YP_421407.1/ YP_421409.1/ YP_421410.1/ chromosome magneticum AMB 1 5060 5682 731 2912 1830 4398 uid58527 YP_421406.1/ 732 Marinobacter aquaeolei 627682 647741 YP_957843.1/ YP_957844.1/ YP_957845.1/ YP_957849.1/ YP_957852.1/ YP_957853.1/ chromosome VT8 uid59419 5061 5683 733 2913 1831 4399 Methanobrevibacter 1795596 1818644 YP_001274322.1/ YP_001274323.1/ YP_001274324.1/ YP_001274316.1/ YP_001274326.1/ YP_001274327.1/ One chromosome smithii ATCC 35061 5015 5632 681 2840 1780 6165 complete uid58827 YP_001274317.1/ methylase 2841 and 5 YP_001274318.1/ truncated. 2842 YP_001274319.1/ 2843 YP_001274320.1/ 2844 YP_001274321.1/ 2845 Methanosarcina 2919156 2939722 Missing NP_617281.1/ NP_617280.1/ NP_617279.1/ NP_617273.1/ NP_617272.1/ chromosome acetivorans C2A 5636 685 2852 1784 4358 uid57879 Methanosarcina mazei 193892 214864 Missing NP_632177.1/ NP_632178.1/ NP_632180.1/ NP_632187.1/ NP_632188.1/ chromosome Gol uid57893 5637 686 2853 1785 4359 Methanospirillum 1213818 1228530 YP_502553.1/ YP_502552.1/ YP_502551.1/ YP_502549.1/ YP_502546.1/ YP_502545.1/ chromosome hungatei JF 1 uid58181 5017 5638 687 2854 1786 4360 Methanospirillum 2137148 2153998 Missing YP_503327.1/ YP_503328.1/ YP_503329.1/ YP_503337.1/ YP_503338.1/ chromosome hungatei JF 1 uid58181 5639 688 2855 1787 4361 Microlunatus 4106044 4121697 YP_004574223.1/ YP_004574224.1/ YP_004574225.1/ YP_004574226.1/ YP_004574231.1/ YP_004574232.1/ methylase chromosome phosphovorus NM 1 5018 5640 689 2856 1788 4362 split into uid68055 YP_004574227.1/ three. 2857 YP_004574229.1/ 2858 Moorella thermoacetica 2331992 2347051 YP_431074.1/ YP_431073.1/ YP_431072.1/ YP_431068.1 YP_431067.1/ YP_431066.1/ Split chromosome ATCC 39073 uid58051 5019 5641 690 YP_431071.1 1789 4363 methylase. Nostoc punctiforme PCC 6986741 7000722 YP_001868910.1/ YP_001868903.1/ YP_001868904.1/ YP_001868905.1/ YP_001868908.1/ YP_001868909.1/ chromosome 73102 uid57767 5062 5684 734 2914 1832 4400 Parvularcula 1068561 1081391 YP_003854347.1/ YP_003854346.1/ YP_003854345.1/ YP_003854344.1/ YP_003854343.1/ YP_003854342.1/ chromosome bermudensis HTCC2503 5020 5642 691 2861 1790 4364 uid51641 Pelobacter propionicus 2459986 2475529 YP_901956.1/ YP_901955.1/ YP_901954.1/ YP_901953.1/ YP_901949.1/ YP_901948.1/ chromosome DSM 2379 uid58255 5022 5644 693 2864 1792 4366 Pelodictyon 2122302 2136557 YP_002018860.1/ YP_002018861.1/ YP_002018862.1/ YP_002018863.1/ YP_002018865.1/ YP_002018866.1/ chromosome phaeoclathratiforme BU 5023 5645 694 2865 1793 4367 1 uid58173 Photorhabdus 470261 481491 YP_003039266.1/ YP_003039267.1/ YP_003039268.1/ YP_003039269.1/ YP_003039270.1/ Missing chromosome asymbiotica ATCC 5024 5646 695 2866 1794 43949 uid59243 Polaromonas JS666 120587 142415 YP_551792.1/ YP_551793.1/ YP_551794.1/ YP_551797.1/ YP_551801.1/ YP_551802.1/ plasmid uid58207 5025 5647 696 2867 1795 4368 Pseudomonas 940354 955543 YP_004351894.1/ YP_004351895.1/ YP_004351896.1/ YP_004351897.1/ YP_004351899.1/ YP_004351900.1/ chromosome brassicacearum NFM421 5026 5648 697 2868 1796 4369 uid66303 Psychrobacter 1973414 1984296 YP_580872.1/ YP_580871.1/ YP_580870.1/ YP_580869.1/ YP_580868.1/ Missing chromosome cryohalolentis K5 5028 5650 699 2871 1798 uid58373 Rhodobacter sphaeroides 628478 642432 YP_001169907.1/ YP_001169906.1/ Rsph17025_3734/ YP_001169903.1/ YP_001169902.1/ YP_001169901.1/ plasmid ATCC 17025 uid58451 5063 5685 615 2915 1833 4401 Rhodococcus 3099110 3111990 YP_002766345.1/ YP_002766344.1/ YP_002766343.1/ YP_002766342.1/ YP_002766341.1/ YP_002766340.1/ chromosome erythropolis PR4 5029 5651 700 2872 1799 4371 uid59019 Rhodopseudomonas 1186657 1200147 YP_001990139.1/ YP_001990140.1/ YP_001990141.1/ YP_001990142.1/ YP_001990143.1/ YP_001990144.1/ chromosome palustris TIE 1 uid58995 5030 5652 701 2873 1800 4372 Runella slithyformis 4922401 4938169 YP_004657746.1/ YP_004657745.1/ YP_004657744.1/ YP_004657743.1/ YP_004657738.1/ YP_004657737.1/ chromosome DSM 19594 uid68317 5064 5686 735 2916 1834 4402 Saccharophagus 3482582 3496946 YP_528231.1/ YP_528230.1/ YP_528229.1/ YP_528228.1/ YP_528225.1/ YP_528224.1/ chromosome degradans 2 40 uid57921 5031 5653 702 2874 1801 4373 Salmonella enterica 4736680 4751839 NP_463357.1/ NP_463356.1/ NP_463355.1/ NP_463354.1/ NP_463351.1/ NP_463350.1/ chromosome serovar Typhimurium 5034 5656 705 2877 1804 4376 LT2 uid57799 Selenomonas sputigena 820667 833330 YP_004413153.1/ YP_004413154.1/ YP_004413155.1/ YP_004413156.1/ YP_004413158.1/ Missing chromosome ATCC 35185 uid66335 5040 5662 711 2883 1810 Shewanella ANA 3 2138213 2155059 YP_869455.1/ YP_869456.1/ YP_869457.1/ YP_869458.1/ YP_869460.1/ Missing chromosome uid58347 5041 5663 712 2884 1811 Shewanella MR 4 2013300 2030132 YP_733838.1/ YP_733839.1/ YP_733840.1/ YP_733841.1/ YP_733843.1/ Missing chromosome uid58345 5042 5664 713 2885 1812 Slackia 349611 366195 YP_003142715.1/ YP_003142714.1/ YP_003142713.1/ YP_003142710.1/ YP_003142712.1/ YP_003142708.1/ Two full chromosome heliotrinireducens DSM 5043 5665 714 2886 1813 4382 length 20476 uid59051 YP_003142711.1/ methylases. 2887 Spirosoma linguale DSM 5893753 5908211 YP_003389619.1/ YP_003389620.1/ YP_003389621.1/ YP_003389624.1/ YP_003389625.1/ YP_003389626.1/ chromosome 74 uid43413 5044 5666 715 2888 1814 4383 Sulfuricurvum kujiense 1691724 1708882 YP_004060547.1/ YP_004060548.1/ YP_004060549.1/ YP_004060550.1 YP_004060557.1/ YP_004060558.1/ Split chromosome DSM 16994 uid60789 5045 5667 716 YP_004060555.1 1815 4384 methylase. Syntrophomonas wolfei 2823947 2838578 YP_755156.1/ YP_755155.1/ YP_755154.1/ YP_755151.1 YP_755149.1/ YP_755148.1/ One chromosome Goettingen uid58179 5048 5670 719 YP_755153.1 1818 4387 complete methylase and one truncated. Syntrophomonas wolfei 1119627 1137079 YP_753673.1/ YP_753674.1/ YP_753675.1/ YP_753676.1/ YP_753683.1/ YP_753684.1/ Methylase chromosome Goettingen uid58179 5047 5669 718 2893 1817 4386 split into YP_753679.1/ three. 2894 YP_753682.1/ 2895 Syntrophus 932423 951906 YP_460955.1/ YP_460954.1/ YP_460953.1/ YP_460949.1/ YP_460946.1/ YP_460945.1/ chromosome aciditrophicus SB 5049 5671 720 2899 1819 4388 uid58539 Tepidanaerobacter Re1 564518 579372 YP_004460026.1/ YP_004460027.1/ YP_004460028.1/ YP_004460030.1/ YP_004460031.1/ YP_004460032.1/ chromosome uid66873 5051 5673 722 2902 1821 4390 Thauera MZ1T uid58987 329333 347549 YP_002353978.1/ YP_002353979.1/ YP_002353980.1/ YP_002353983.1/ YP_002353988.1/ YP_002353989.1/ chromosome 5052 5674 723 2903 1822 4391 Thermoanaerobacterium 483298 496318 YP_003851149.1/ YP_003851150.1/ YP_003851151.1/ YP_003851152.1/ YP_003851153.1/ YP_003851154.1/ chromosome thermosaccharolyticum 5054 5676 725 2905 1824 4393 DSM 571 uid51639 Vibrio cholerae MJ 1236 3021296 3036340 YP_002879448.1/ YP_002879447.1/ YP_002879446.1/ YP_002879444.1/ YP_002879443.1/ YP_002879442.1/ chromosome uid59387 5057 5679 728 2909 1827 4395 Zymomonas mobilis 1590155 1602970 YP_003226496.1/ YP_003226497.1/ YP_003226498.1/ YP_003226499.1/ YP_003226500.1/ YP_003226501.1/ chromosome NCIMB 11163 uid41019 5059 5681 730 2911 1829 4397 Thioalkalivibrio sp. 1195163 1212198 YP_003460374.1/ YP_003460375.1/ TK90_1129 (no YP_003460378.1/ YP_003460381.1/ YP_003460382.1/ chromosome K90mix 6225 6227 accession)/ 6231 6233 6235 6229 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 3 BREX type 5 systems Genomic Genomic Start End Organism Point Point BrxA* BrxB* BrxC/PglY* PglX* Haloarcula 401474 426563 YP_004786064.1/ YP_004786062.1/ YP_004786061.1/ YP_004786055.1/ hispanica 5068 5691 744 2922 ATCC 33960 YP_004786063.1/ YP_004786058.1/ uid72475 745 2923 Halobacterium 213889 239329 YP_001690762.1/ YP_001690760.1/ YP_001690761.1/ YP_001690755.1/ salinarum R1 5066 5689 741 2919 uid61571 YP_001690759.1/ 740 halophilic 245839 265731 YP_004809887.1/ YP_004809889.1/ YP_004809888.1/ YP_004809893.1/ archaeon 5069 5692 746 2924 DL31 YP_004809890.1/ uid72619 747 Halopiger 275443 305939 YP_004595482.1/ YP_004595484.1/ YP_004595483.1/ YP_004595494.1/ xanaduensis 5065 5687 736 2917 SH 6 YP_004595485.1/ uid68105 737 Halorubrum 421884 442192 YP_002564617.1/ YP_002564615.1/ YP_002564616.1/ YP_002564611.1/ lacusprofundi 5067 5690 743 2920 ATCC 49239 YP_002564614.1/ uid58807 742 Halorhabdus 1919731 1943689 257052977/ YP_003130812.1/ YP_003130811.1/ YP_003130818.1/ utahensis 6171 5688 738 2918 DSM 12940 YP_003130813.1/ uid59189 739 Methylase Replicon Organism PglZ* BrxHII* State Type Haloarcula YP_004786056.1/ YP_004786054.1/ Two full chromosome hispanica 1839 3501 length ATCC 33960 methylases. uid72475 Halobacterium YP_001690753.1/ plasmid salinarum R1 1837 uid61571 halophilic YP_004809895.1/ YP_004809896.1/ plasmid archaeon 1840 3502 DL31 uid72619 Halopiger YP_004595497.1/ YP_004595499.1/ chromosome xanaduensis 1835 3498 SH 6 uid68105 Halorubrum YP_002564610.1/ YP_002564609.1/ chromosome lacusprofundi 1838 3500 ATCC 49239 uid58807 Halorhabdus YP_003130820.1/ YP_003130822.1/ chromosome utahensis 1836 3499 DSM 12940 uid59189 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 4 BREX type 6 systems Genomic Genomic Start End Organism Point Point BrxE* BrxA* BrxB* BrxC/PglY* Anaeromyxobacter 1284321 1301040 YP_002491563.1/ YP_002491564.1/ YP_002491565.1/ YP_002491566.1/ dehalogenans 2CP 6038 5072 5695 751 1 uid58989 Haliangium 1611313 1628687 YP_003265686.1/ YP_003265687.1/ YP_003265688.1/ YP_003265689.1/ ochraceum DSM 6040 5074 5697 753 14365 uid41425 Haliangium 798493 815906 YP_003265127.1/ YP_003265128.1/ YP_003265129.1/ YP_003265130.1/ ochraceum DSM 6039 5073 5696 752 14365 uid41425 Planctomyces 3979304 3995634 YP_003631101.1/ YP_003631100.1/ YP_003631099.1/ YP_003631098.1/ limnophilus DSM 6037 5071 5694 750 3776 uid48643 Organism PglX* PglZ* BrxD* BrxHI* Anaeromyxobacter YP_002491567.1/ YP_002491568.1/ YP_002491570.1/ YP_002491571.1/ dehalogenans 2CP 2928 1843 4442 3623 1 uid58989 Haliangium YP_003265690.1/ YP_003265691.1/ YP_003265692.1/ YP_003265693.1/ ochraceum DSM 2930 1845 4444 3625 14365 uid41425 Haliangium YP_003265131.1/ YP_003265132.1/ YP_003265133.1/ YP_003265134.1/ ochraceum DSM 2929 1844 4443 3624 14365 uid41425 Planctomyces YP_003631097.1/ YP_003631096.1/ YP_003631095.1/ YP_003631094.1/ limnophilus DSM 2927 1842 4441 3622 3776 uid48643 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 5 BREX type 3 systems Genomic Genomic Start End Organism Point Point BrxF* BrxC/PglY* PglXI* BrxHII* PglZ* BrxA* Acidothermus 895934 911157 YP_872570.1/ YP_872571.1/ YP_872573.1/ YP_872575.1/ YP_872576.1/ YP_872577.1/ cellulolyticus 11B 5980 755 3344 3504 1847 5076 uid58501 Parvibaculum 1304574 1320948 YP_001412459.1/ YP_001412458.1/ YP_001412455.1/ YP_001412454.1/ YP_001412453.1/ YP_001412452.1/ lavamentivorans DS 1 5992 769 3360 3515 1860 5089 uid58739 Parvibaculum 3796620 3812997 YP_001414809.1/ YP_001414810.1/ YP_001414813.1/ YP_001414814.1/ YP_001414815.1/ YP_001414816.1/ lavamentivorans DS 1 5993 770 3361 3516 1861 5090 uid58739 Chloroflexus aggregans 1376256 1391910 YP_002462464.1/ YP_002462466.1/ YP_002462465.1/ YP_002462469.1/ YP_002462470.1/ YP_002462471.1/ DSM 9485 uid58621 5983 759 3348 3507 1851 5080 Desulfovibrio 1822100 1838149 YP_004121411.1/ YP_004121412.1/ YP_004121414.1/ YP_004121416.1/ YP_004121417.1/ YP_004121418.1/ aespoeensis Aspo 2 5984 760 3350 3508 1852 5081 uid42613 Methanosalsum zhilinae 1398870 1421705 YP_004616377.1/ YP_004616376.1/ YP_004616375.1/ YP_004616371.1/ YP_004616370.1/ YP_004616369.1/ DSM 4017 uid68249 5987 764 3353 3510 1855 5084 Caldicellulosiruptor 671655 683698 YP_004025779.1/ YP_004025780.1/ YP_004025781.1/ Calkr_0625/ YP_004025782.1/ YP_004025783.1/ kristjanssonii 177R1B 5982 758 3347 6173 1850 5079 uid60393 Pelotomaculum 698030 719373 YP_001211256.1/ YP_001211265.1/ YP_001211266.1/ YP_001211267.1/ / thermopropionicum SI 616 3403 3493 1715 uid58877 Thermoanaerobacter 974096 986151 YP_004185913.1/ YP_004185914.1/ YP_004185915.1/ YP_004185916.1/ YP_004185917.1/ YP_004185918.1/ brockii finnii Ako 1 5999 777 3367 3519 1867 5096 uid55639 Thermoanaerobacter 981903 993958 YP_001664916.1/ YP_001664917.1/ YP_001664918.1/ YP_001664919.1/ YP_001664920.1/ YP_001664921.1/ pseudethanolicus ATCC 6001 779 3369 3520 1869 5098 33223 uid58339 Thermoanaerobacterium 1036830 1048931 YP_004470679.1/ YP_004470680.1/ YP_004470681.1/ YP_004470682.1/ YP_004470683.1/ YP_004470684.1/ xylanolyticum LX 11 6004 782 3372 3523 1872 5101 uid63163 Thermoanaerobacter 1369768 1379419 YP_003477173.1/ YP_003477172.1/ YP_003477171.1/ Missing YP_003477169.1/ YP_003477168.1/ italicus Ab9 uid46241 6000 778 3368 1868 5097 Syntrophothermus 1307417 1316966 YP_003702612.1/ YP_003702611.1/ YP_003702610.1/ Missing YP_003702609.1/ YP_003702608.1/ lipocalidus DSM 12680 5995 772 3363 1863 5092 uid49527 Acetohalobium 1465881 1481230 YP_003827965.1/ YP_003827964.1/ YP_003827963.1/ Missing YP_003827962.1/ YP_003827961/ arabaticum DSM 5501 5979 754 3343 1846 5075 uid51423 Dichelobacter nodosus 185804 200163 YP_001209111.1/ YP_001209110.1/ YP_001209109.1/ YP_001209106.1/ YP_001209105.1/ YP_001209104.1/ VCS1703A uid57643 5985 761 3351 3509 1853 5082 Nitrosococcus oceani 53559 70307 YP_342127.1/ YP_342128.1/ YP_342131.1/ YP_342135.1/ YP_342136.1/ YP_342137.1/ ATCC 19707 uid58403 5990 767 3358 3513 1858 5087 Nitrosococcus watsonii 43703 58760 YP_003759356.1/ YP_003759357.1/ YP_003759359.1/ YP_003759363.1/ YP_003759364.1/ YP_003759365.1/ C 113 uid50331 5991 768 3359 3514 1859 5088 Methylacidiphilum 317802 333912 YP_001938984.1/ YP_001938983.1/ YP_001938982.1/ YP_001938980.1/ YP_001938979.1/ YP_001938978.1/ infernorum V4 5988 765 3356 3511 1856 5085 uid59161 Thermanaerovibrio 1551826 1568220 YP_003318004.1/ YP_003318003.1/ YP_003318001.1/ YP_003317999.1/ YP.003317998.1/ YP_003317997.1/ acidaminovorans DSM 5998 776 3366 3518 1866 5095 6589 uid41925 Planctomyces 1319343 1335543 YP_004268781.1/ YP_004268780.1/ YP_004268778.1/ YP_004268777.1/ YP_004268776.1/ YP_004268775.1/ brasiliensis DSM 5305 5994 771 3362 3517 1862 5091 uid60583 Tepidanaerobacter Re1 1934428 1943118 YP_004461309.1/ YP_004461308.1/ YP_004461307.1/ missing YP_004461306.1/ YP_004461305.1/ uid66873 5997 775 3365 1865 5096 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 6 BREX type 2 systems Genomic Genomic Start End Organism Point Point PglW* PglX* PglY* PglZ* BrxD* BrxHI* Candidatus 3869550 3890263 YP_003168573.1/ YP_003168572.1/ YP_003168567.1/ YP_003168566.1/ YP_003168565.1/ YP_003168564.1/ Accumulibacter 6092 2932 784 1874 4445 3596 phosphatis clade IIA UW 1 uid59207 Corynebacterium 1913373 1933608 YP_004760098.1/ YP_004760099.1/ YP_004760100.1/ YP_004760101.1/ YP_004760102.1/ YP_004760103.1/ variabile DSM 44702 6094 2934 787 1876 4446 3598 uid62003 Frankia CcI3 3489708 3507585 YP_482039.1/ YP_482040.1/ YP_482041.1/ YP_482042.1/ YP_482043.1/ YP_482044.1/ uid58397 6095 2935 788 1877 4447 3599 Frankia EuI1c 6951904 6971263 YP_004019519.1/ YP_004019520.1/ YP_004019521.1/ YP_004019522.1/ YP_004019523.1/ YP_004019524.1/ uid42615 6096 2936 789 1878 4448 3600 Hahella chejuensis 3587257 3606877 YP_434642.1/ YP_434639.1/ YP_434638.1/ YP_434637.1/ YP_434636.1/ YP_434635.1/ KCTC 2396 uid58483 6114 2953 806 1894 4462 3615 Haliangium 1565170 1582937 YP_003265661.1/ YP_003265662.1/ YP_003265663.1/ YP_003265664.1/ YP_003265665.1/ YP_003265666.1/ ochraceum DSM 6097 2937 790 1879 791 3601 14365 uid41425 Microlunatus 3075705 3093432 YP_004573280.1/ YP_004573279.1/ YP_004573278.1/ YP_004573277.1/ YP_004573276.1/ YP_004573275.1/ phosphovorus NM 1 6098 2938 792 1880 4449 3602 uid68055 Micromonospora 1329830 1350410 YP_003834429.1/ YP_003834430.1/ YP_003834433.1/ YP_003834434.1/ YP_003834435.1/ YP_003834436.1/ aurantiaca ATCC 6099 2939 793 1881 4450 3603 27029 uid42501 Mycobacterium 3386977 3404461 YP_001134469.1/ YP_001134468.1/ YP_001134467.1/ YP_001134466.1/ YP_001134465.1/ YP_001134464.1/ gilvum PYR GCK 6100 2940 794 1882 4451 3604 uid59421 Polaromonas 170793 191771 YP_973309.1/ YP_973307.1/ YP_973304.1/ YP_973303.1/ YP_973302.1/ YP_973301.1/ naphthalenivorans 6102 2942 796 1884 4453 3606 CJ2 uid58273 Saccharopolyspora 5714083 5716377 YP_001107302.1/ YP_001107301.1/ YP_001107300.1/ YP_001107299.1/ YP_001107298.1/ YP_001107297.1/ erythraea NRRL 2338 6104 2945 798 1886 4455 3608 uid62947 Sorangium 1.10E+07 10706714 YP_001618324.1/ YP_001618325.1/ YP_001618331.1/ YP_001618334.1/ YP_001618335.1/ YP_001618336.1/ cellulosum So ce 56 6107 2948 801 1889 4458 3611 uid61629 Streptomyces 7348537 7376403 NP_630703.1/ NP_733709.1/ NP_630711.1/ NP_630712.1/ NP_630715.1/ NP_630716.1/ coelicolor A3 2 6110 2949 802 1890 4459 3612 uid57801 Streptomyces griseus 1877109 1900853 YP_001823112.1/ YP_001823113.1/ YP_001823118.1/ YP_001823119.1/ YP_001823122.1/ YP_001823123.1/ NBRC 13350 6111 2950 803 1891 4460 3613 uid58983 Thermobifida fusca 810381 830646 YP_288762.1/ Rsph17025_3734/ YP_288770.1/ YP_288771.1/ YP_288772.1/ YP_288773.1/ YX uid57703 6112 615 804 1892 4461 3614 Burkholderia 131918 145741 YP_440673.1/ YP_440674.1/ YP_440675.1/ YP_440676.1/ Missing Missing thailandensis E264 6091 2931 783 1873 uid58081 Thermobispora 1764882 779583 YP_003652152.1/ YP_003652153.1/ YP_003652154.1/ YP_003652155.1/ Missing Missing bispora DSM 43833 6113 2952 805 1893 uid48999 Saccharomonospora 508003 530821 YP_003132413.1/ YP_003132422.1/ YP_003132423.1/ YP_003132424.1/ YP_003132425.1/ YP_003132426.1/ viridis DSM 43017 6103 2944 797 1885 4454 3607 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 7 BREX type 4 systems Genomic Genomic Start End Organism Point Point BrxP* BrxC/PglY* PglZ* BrxL* Candidatus 837185 848109 YP_001716949.1/ YP_001716950.1/ YP_001716952.1/ YP_001716953.1/ Desulforudis 3431 809 1897 4405 audaxviator MP104C uid59067 Coprothermobacter 1373789 1384972 YP_002247820.1/ YP_002247818.1/ YP_002247817.1/ YP_002247816.1/ proteolyticus DSM 3433 810 1898 4406 5265 uid59253 YP_002247819.1/ 3432 Denitrovibrio 583467 593589 YP_003503325.1/ YP_003503326.1/ YP_003503327.1/ YP_003503328.1/ acetiphilus DSM 3435 813 1900 4408 12809 uid46657 Geobacter M21 937933 948070 YP_003020622.1/ YP_003020623.1/ YP_003020624.1/ YP_003020625.1/ uid59037 3437 815 1902 4410 Prevotella 414128 424371 YP_004328097.1/ YP_004328099.1/ YP_004328100.1/ YP_004328101.1/ denticola F0289 3438 816 1903 4411 uid65091 Thermomicrobium 91162 104624 YP_002523385.1/ YP_002523389.1/ YP_002523390.1/ roseum DSM 5159 614 1714 4299 uid59341 Thermotoga 1747153 1756730 YP_001245328.1/ YP_001245329.1/ YP_001245330.1/ YP_001245331.1/ petrophila RKU 1 3439 817 1904 4412 uid58655 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 8 Summary of distribution of BREX types across genomes Taxon BREX BREX BREX BREX BREX BREX ID Organism Kingdom #1 #5 #6 #3 #2 #4 Comments 1 882 Desulfovibrio vulgaris Bacteria X Hildenborough uid57645 2 56780 Syntrophus aciditrophicus Bacteria X SB uid58539 3 60480 Shewanella MR 4 uid58345 Bacteria X 4 62140 Acidiphilium multivorum Bacteria X AIU301 uid63345 5 63737 Nostoc punctiforme PCC Bacteria X 73102 uid57767 6 76114 Aromatoleum aromaticum Bacteria X EbN1 uid58231 7 85643 Thauera MZ1T uid58987 Bacteria X 8 94122 Shewanella ANA 3 Bacteria X uid58347 9 99287 Salmonella enterica serovar Bacteria X Typhimurium LT2 uid57799 10 138119 Desulfitobacterium Bacteria X hafniense Y51 uid58605 11 188937 Methanosarcina acetivorans Archaea X C2A uid57879 12 192952 Methanosarcina mazei Go1 Archaea X uid57893 13 203122 Saccharophagus degradans Bacteria X 2 40 uid57921 14 234621 Rhodococcus erythropolis Bacteria X PR4 uid59019 15 243231 Geobacter sulfurreducens Bacteria X PCA uid57743 16 246194 Carboxydothermus Bacteria X hydrogenoformans Z 2901 uid57821 17 262543 Exiguobacterium sibiricum Bacteria X 255 15 uid58053 18 264732 Moorella thermoacetica Bacteria X ATCC 39073 uid58051 19 269482 Burkholderia vietnamiensis Bacteria X G4 uid58075 20 290397 Anaeromyxobacter Bacteria X dehalogenans 2CP C uid58135 21 296591 Polaromonas JS666 Bacteria X uid58207 22 311424 Dehalococcoides VS Bacteria X uid42393 23 314225 Erythrobacter litoralis Bacteria X HTCC2594 uid58299 24 314260 Parvularcula bermudensis Bacteria X HTCC2503 uid51641 25 323259 Methanospirillum hungatei Archaea XX This genome contains JF 1 uid58181 two BREX systems of type 1 26 324925 Pelodictyon Bacteria X phaeoclathratiforme BU 1 uid58173 27 331112 Escherichia coli HS Bacteria X uid58393 28 331678 Chlorobium Bacteria X phaeobacteroides BS1 uid58131 29 335284 Psychrobacter Bacteria X cryohalolentis K5 uid58373 30 335541 Syntrophomonas wolfei Bacteria XX This genome contains Goettingen uid58179 two BREX systems of type 1 31 338966 Pelobacter propionicus Bacteria X DSM 2379 uid58255 32 342108 Magnetospirillum Bacteria X magneticum AMB 1 uid58527 33 349102 Rhodobacter sphaeroides Bacteria X ATCC 17025 uid58451 34 351348 Marinobacter aquaeolei Bacteria X VT8 uid59419 35 395494 Gallionella Bacteria XX This genome contains capsiferriformans ES 2 two BREX systems of uid51505 type 1 36 395960 Rhodopseudomonas Bacteria X palustris TIE 1 uid58995 37 395962 Cyanothece PCC 8802 Bacteria X uid59143 38 420247 Methanobrevibacter smithii Archaea X ATCC 35061 uid58827 39 471223 Geobacillus WCH70 Bacteria X uid59045 40 471855 Slackia heliotrinireducens Bacteria X DSM 20476 uid59051 41 491077 Lactobacillus reuteri Bacteria X SD2112 uid55357 42 498216 Lactobacillus casei Zhang Bacteria X uid50673 43 499177 Clostridium sticklandii Bacteria X DSM 519 uid59585 44 504472 Spirosoma linguale DSM Bacteria X 74 uid43413 45 509173 Acinetobacter baumannii Bacteria X AYE uid61637 46 525897 Desulfomicrobium Bacteria X baculatum DSM 4028 uid59217 47 546271 Selenomonas sputigena Bacteria X ATCC 35185 uid66335 48 552811 Dehalogenimonas Bacteria X lykanthroporepellens BL DC 9 uid48131 49 553480 Photorhabdus asymbiotica Bacteria X ATCC 43949 uid59243 50 568703 Lactobacillus rhamnosus Bacteria X GG uid59313 51 573370 Desulfovibrio magneticus Bacteria X RS 1 uid59309 52 580327 Thermoanaerobacterium Bacteria X thermosaccharolyticum DSM 571 uid51639 53 585054 Escherichia fergusonii Bacteria X ATCC 35469 uid59375 54 585396 Escherichia coli O111 H Bacteria X 11128 uid41023 55 589865 Desulfurivibrio alkaliphilus Bacteria X AHT2 uid49487 56 593588 Vibrio cholerae MJ 1236 Bacteria X uid59387 57 610130 Clostridium Bacteria XX This genome contains saccharolyticum WM1 two BREX systems of uid51419 type 1 58 622759 Zymomonas mobilis Bacteria X NCIMB 11163 uid41019 59 633699 Lactobacillus johnsonii Bacteria X FI9785 uid41735 60 634499 Erwinia pyrifoliae Ep1 96 Bacteria X uid40659 61 640510 Burkholderia CCGE1001 Bacteria X uid42975 62 709032 Sulfuricurvum kujiense Bacteria X DSM 16994 uid60789 63 743299 Acidithiobacillus Bacteria X ferrivorans SS3 uid67387 64 748727 Clostridium ljungdahlii Bacteria X DSM 13528 uid50583 65 760192 Haliscomenobacter Bacteria X hydrossis DSM 1100 uid66777 66 761193 Runella slithyformis DSM Bacteria X 19594 uid68317 67 762051 Leuconostoc kimchii Bacteria X IMSNU 11154 uid48589 68 768670 Calditerrivibrio Bacteria X nitroreducens DSM 19672 uid60821 69 994484 Pseudomonas Bacteria X brassicacearum NFM421 uid66303 70 999541 Burkholderia gladioli BSR3 Bacteria X uid66301 71 1032480 Microlunatus phosphovorus Bacteria X X This genome contains NM 1 uid68055 two BREX systems (types 1 and 2) 72 1042156 Clostridium SY8519 Bacteria X uid68705 73 1042878 Cupriavidus necator N 1 Bacteria X uid68689 74 1209989 Tepidanaerobacter Re1 Bacteria X X This genome contains uid66873 two BREX systems (types 1 and 3) 75 416348 Halorubrum lacusprofundi Archaea X ATCC 49239 uid58807 76 478009 Halobacterium salinarum Archaea X R1 uid61571 77 519442 Halorhabdus utahensis Archaea X DSM 12940 uid59189 78 634497 Haloarcula hispanica ATCC Archaea X 33960 uid72475 79 756883 halophilic archaeon DL31 Archaea X uid72619 80 797210 Halopiger xanaduensis SH Archaea X 6 uid68105 81 455488 Anaeromyxobacter Bacteria X dehalogenaris 2CP 1 uid58989 82 502025 Haliangium ochraceum Bacteria XX X This genome contains DSM 14365 uid41425 three BREX systems (two types 6 and one of type 2) 83 521674 Planctomyces limnophilus Bacteria X DSM 3776 uid48643 84 105559 Nitrosococcus watsonii C Bacteria X 113 uid50331 85 246195 Dichelobacter nodosus Bacteria X VCS1703A uid57643 86 323261 Nitrosococcus oceani Bacteria X ATCC 19707 uid58403 87 326427 Chloroflexus aggregans Bacteria X DSM 9485 uid58621 88 340099 Thermoanaerobacter Bacteria X pseudethanolicus ATCC 33223 uid58339 89 351607 Acidothermus cellulolyticus Bacteria X 11B uid58501 90 370438 Pelotomaculum Bacteria X thermopropionicum SI uid58877 91 402881 Parvibaculum Bacteria XX This genome contains lavamentivorans DS 1 two BREX systems of uid58739 type 3 92 481448 Methylacidiphilum Bacteria X infernorum V4 uid59161 93 509193 Thermoanaerobacter brockii Bacteria X finnii Ako 1 uid55639 94 525903 Thermanaerovibrio Bacteria X acidaminovorans DSM 6589 uid41925 95 574087 Acetohalobium arabaticum Bacteria X DSM 5501 uid51423 96 580331 Thermoanaerobacter Bacteria X italicus Ab9 uid46241 97 632335 Caldicellulosiruptor Bacteria X kristjanssonii 177R1B uid60393 98 643562 Desulfovibrio aespoeensis Bacteria X Aspo 2 uid42613 99 643648 Syntrophothermus Bacteria X lipocalidus DSM 12680 uid49527 100 679901 Methanosalsum zhilinae Archaea X DSM 4017 uid68249 101 756272 Planctomyces brasiliensis Bacteria X DSM 5305 uid60583 102 858215 Thermoanaerobacterium Bacteria X xylanolyticum LX 11 uid63163 103 100226 Streptomyces coelicolor A3 Bacteria X 2 uid57801 104 106370 Frankia CcI3 uid58397 Bacteria X 105 269800 Thermobifida fusca YX Bacteria X uid57703 106 271848 Burkholderia thailandensis Bacteria X E264 uid58081 107 298654 Frankia EuI1c uid42615 Bacteria X 108 349521 Hahella chejuensis KCTC Bacteria X 2396 uid58483 109 350054 Mycobacterium gilvum Bacteria X PYR GCK uid59421 110 365044 Polaromonas Bacteria X naphthalenivorans CJ2 uid58273 111 405948 Saccharopolyspora Bacteria X erythraea NRRL 2338 uid62947 112 448385 Sorangium cellulosum So Bacteria X ce 56 uid61629 113 455632 Streptomyces griseus Bacteria X NBRC 13350 uid58983 114 469371 Thermobispora bispora Bacteria X DSM 43833 uid48999 115 522306 Candidatus Accumulibacter Bacteria X phosphatis clade IIA UW 1 uid59207 116 644283 Micromonospora aurantiaca Bacteria X ATCC 27029 uid42501 117 858619 Corynebacterium variabile Bacteria X DSM 44702 uid62003 118 309798 Coprothermobacter Bacteria X proteolyticus DSM 5265 uid59253 119 309801 Thermomicrobium roseum Bacteria X DSM 5159 uid59341 120 390874 Thermotoga petrophila Bacteria X RKU 1 uid58655 121 443144 Geobacter M21 uid59037 Bacteria X 122 477974 Candidatus Desulforudis Bacteria X audaxviator MP104C uid59067 123 522772 Denitrovibrio acetiphilus Bacteria X DSM 12809 uid46657 124 767031 Prevotella denticola F0289 Bacteria X uid65091 125 396595 Thioalkalivibrio sp. Bacteria X K90mix 126 471857 Saccharomonospora viridis Bacteria X DSM 43017

TABLE 10 BREX type 1 systems Genomic Genomic Start End Organism Point Point brxA* brxB* brxC* pglX* pglZ* brxL* Acidiphilium 497516 512043 326402595/ 326402594/ 326402593/ 326402591/ 326402589/ 326402588/ multivorum AIU301 4953 5570 617 2766 1716 4300 uid63345 326402592/ 2767 Acidithiobacillus 2361379 2377751 344200465/ 344200466/ 344200467/ 344200468/ 344200473/ 344200474/ ferrivorans SS3 4954 5571 618 2768 1717 4301 uid67387 344200470/ 2769 Acinetobacter 53839 68427 169786944/ 169786943/ 169786942/ 169786941/ 169786939/ 169786938/ baumannii AYE 4955 5572 619 2770 1718 4302 uid61637 Alteromonas macleodii 2290017 2302123 407700182/ 407700181/ 407700180/ 407700179/ 407700176/ missing Black Sea 11 4956 5573 620 2771 1719 uid176365 Anaeromyxobacter 3688393 3701032 86159656/ 86159655/ 86159654/ 86159653/ 86159652/ 86159651/ dehalogenans 2CP C 4957 5574 621 2772 1720 4303 uid58135 Aromatoleum 3091305 3115719 56478397/ 56478398/ 56478399/ 56478402/ 56478415/ 56478416/ aromaticum EbN1 4958 5575 622 2773 1721 4304 uid58231 Arthrobacter 73164 86048 403571626/ 403571625/ 403571624/ 403571623/ 403571622/ 403571621/ nitroguajacolicus 4959 5576 623 2774 1722 4305 Rue61a uid174511 Azospirillum lipoferum 149169 163502 374998149/ 374998148/ 374998147/ 374998346/ 374998144/ 374998143/ 4B uid82343 4960 5577 624 2775 1723 4306 Bifidobacterium 1014525 1027987 386867047/ 386867048/ 386867049/ 386867050/ 386867051/ 386867052/ animalis ATCC 25527 4961 5578 625 2776 1724 4307 uid162513 Bordetella parapertussis 553819 571957 410471294/ 410471295/ 410471296/ missing 410471301/ 410471302/ Bpp5 uid177516 4962 5579 626 1725 4308 Burkholderia 3119203 3136704 323527071/ 323527070/ 323527069/ 323527068/ 323527064/ 323527063/ CCGE1001 uid42975 4963 5580 627 2777 1726 4309 Burkholderia gladioli 1574842 1588059 330816346/ 330816347/ 330816348/ 330816349/ 330816350/ 330816351/ BSR3 uid66301 4964 5581 628 2778 1727 4310 Burkholderia 2201159 2216554 134296121/ 134296120/ 134296119/ missing 134296116/ 134296115/ vietnamiensis G4 4965 5582 629 1728 4311 uid58075 Calditerrivibrio 2095967 2108863 313673975/ 313673974/ 313673973/ 313673970/ 313673969/ 313673968/ nitroreducens DSM 4966 5583 630 2779 1729 4312 19672 uid60821 313673972/ 2780 Carboxydothermus 2348822 2361903 78044585/ 78043641/ 78043274/ 78045163/ 78043267/ 78044476/ hydrogenoformans Z 4967 5584 631 2781 1730 4313 2901 uid57821 Chlorobium 1811539 1826075 189500612/ 189500611/ 189500610/ 189500607/ 189500606/ 189500605/ phaeobacteroides BS1 4968 5585 632 2782 1731 4314 uid58131 Clostridium clariflavum 253005 267426 374294724/ 374294725/ 374294726/ 374294727/ 374294728/ 374294730/ DSM 19732 uid82345 4969 5586 633 2783 1732 4315 Clostridium clariflavum 3398998 3420023 374297215/ 374297214/ 374297213/ 374297205/ 374297210/ 374297209/ DSM 19732 uid82345 4970 5587 634 2784 1733 4316 374297212/ 2785 Clostridium ljungdahlii 3566678 3580351 300856437/ 300856436/ 300856435/ 300856434/ 300856433/ 300856431/ DSM 13528 uid50583 4971 5588 635 2786 1734 4317 Clostridium 3014507 3029581 302387139/ 302387138/ 302387137/ 302387135/ 302387132/ 302387131/ saccharolyticum WM1 4972 5589 636 2787 1735 4318 uid51419 Clostridium 3993471 4020337 302388017/ 302388016/ 302388015/ 302388014/ 302388010/ 302388009/ saccharolyticum WM1 4973 5590 637 2788 1736 4319 uid51419 302388025/ 2789 Clostridium sticklandii 1172754 1188589 310658338/ 310658339/ 310658340/ 310658341/ 310658344/ 310658345/ DSM 519 uid59585 4974 5591 638 2790 1737 4320 Clostridium SY8519 1975332 1994115 339442901/ 339442902/ 339442903/ 339442905/ 339442908/ 339442909/ uid68705 4975 5592 639 2791 1738 4321 Cupriavidus necator N 1 3467556 3485436 339327422/ 339327421/ 339327420/ 339327417/ 339327415/ 339327414/ uid68689 4976 5593 640 2792 1739 4322 Cyanothece PCC 8802 2210337 2225482 missing 257060039/ 257060038/ 257060035/ 257060032/ 257060031/ uid59143 5594 641 2793 1740 4323 257060036/ 2794 Dehalococcoides VS 231698 257903 270307694/ missing 270307693/ 270307692/ 270307691/ 270307690/ uid42393 4977 642 2795 1741 4324 Dehalogenimonas 1578285 1603624 300088705/ 300088704/ 300088703/ 300088702/ 300088701/ 300088700/ lykanthroporepellens 4978 5595 643 2796 1742 4325 BL DC 9 uid48131 Desulfitobacterium 783463 806836 89893421/ missing 89893422/ 89893423/ 89893425/ 89893426/ hafniense Y51 4979 644 2797 1743 4326 uid58605 Desulfobacula toluolica 3358272 3374466 408420315/ 408420314/ 408420313/ 408420312/ 408420308/ 408420307/ Tol2 uid175777 4980 5596 645 2798 1744 4327 Desulfomicrobium 1457030 1471353 256829115/ 256829114/ 256829113/ 256829112/ 256829110/ 256829109/ baculatum DSM 4028 4981 5597 646 2799 1745 4328 uid59217 Desulfosporosinus 360901 375372 402570959/ 402570960/ 402570961/ 402570962/ 402570964/ 402570965/ meridiei DSM 13257 4982 5598 647 2800 1746 4329 uid75097 Desulfovibrio 4071986 4104148 239908253/ 239908252/ 239908251/ 239908250/ 239908248/ 239908247/ magneticus RS 1 4983 5599 648 2801 1747 4330 uid59309 Desulfovibrio vulgaris 2096176 2110453 missing 46580433/ 46580432/ 46580429/ 46580427/ 46580426/ Hildenborough uid57645 5600 649 2802 1748 4331 Desulfovibrio vulgaris 1472132 1487041 387153153/ 387153154/ 387153155/ 387153158/ 387153160/ 387153161/ RCH1 uid161961 4984 5601 650 2803 1749 4332 Desulfurivibrio 2512933 2529324 297570111/ 297570110/ 297570109/ 297570107/ 297570104/ 297570103/ alkaliphilus AHT2 4985 5602 651 2804 1750 4333 uid49487 Enterobacter cloacae 567010 586287 401762044/ 401762043/ 401762042/ 401762037/ 401762036/ 401762035/ ENHKU01 uid172463 4986 5603 652 2805 1751 4334 401762040/ 2806 Erwinia Ejp617 2111476 2142932 385787391/ 385787390/ 385787389/ 385787388/ 385787387/ 385787386/ uid159955 4987 5604 653 2807 1752 4335 Erwinia pyrifoliae DSM 3075305 3107294 387872410/ 387872411/ 387872412/ 387872413/ 387872414/ 387872415/ 12163 uid159693 4988 5605 654 2808 1753 4336 Erwinia pyrifoliae Ep1 3075240 3107369 259909430/ 259909431/ 259909432/ 259909433/ 259909434/ 259909435/ 96 uid40659 4989 5606 655 2809 1754 4337 Erythrobacter litoralis 2963192 2977697 85375770/ 85375772/ 85375773/ 85375776/ 85375777/ 85375778/ HTCC2594 uid58299 4990 5607 656 2810 1755 4338 Escherichia coli clone 4888341 4901877 386637210/ 386637211/ 386637212/ 386637213/ 386637214/ 386637215/ D i14 uid162049 4991 5608 657 2811 1756 4339 Escherichia coli clone 4888341 4901877 386632290/ 386632291/ 386632292/ 386632293/ 386632294/ 386632295/ D i2 uid162047 4992 5609 658 2812 1757 4340 Escherichia coli HS 335287 354284 157159789/ 157159790/ 157159791/ 157159784/ 157159793/ 157159794/ uid58393 4993 5610 659 2813 1758 4341 157159792/ 2814 Escherichia coli O111 5250831 5264277 260871026/ 260871025/ 260871024/ 260871023/ 260871022/ 260871021/ H 11128 uid41023 4994 5611 660 2815 1759 4342 Escherichia fergusonii 13818 28892 218561657/ 218561658/ 218561659/ 218561661/ 218561662/ 218561663/ ATCC 35469 uid59375 4995 5612 661 2816 1760 4343 Exiguobacterium 325862 338929 172056355/ 172056356/ 172056357/ 172056358/ 172056359/ 172056360/ sibiricum 255 15 4996 5613 662 2817 1761 4344 uid58053 Flavobacterium 1185032 1201974 347535923/ 347535922/ 347535921/ 347535920/ 347535914/ 347535913/ branchiophilum FL 15 4997 5614 663 2818 1762 4345 uid73421 Gallionella 927586 949290 302878114/ 302878115/ 302878116/ 302878119/ 302878127/ 302878128/ capsiferriformans ES 2 4998 5615 664 2819 1763 4346 uid51505 Gallionella 1651741 1666045 302878739/ 302878740/ 302878741/ 302878744/ 302878745/ 302878746/ capsifeniformans ES 2 4999 5616 665 2820 1764 4347 uid51505 Geobacillus WCH70 1353239 1367357 239826787/ 239826788/ 239826789/ 239826790/ 239826791/ missing uid59045 5000 5617 666 2821 1765 Geobacter 2316569 2330889 400756604/ 39997207/ 39997206/ 39997205/ 39997204/ 39997203/ sulfurreducens PCA 5001 5618 667 2822 1766 4348 uid57743 Haliscomenobacter 16078 32343 332661910/ 332661909/ 332661908/ 332661907/ 332661904/ 332661903/ hydrossis DSM 1100 5002 5619 668 2823 1767 4349 uid66777 Halobacillus halophilus 4050129 4064174 386716369/ 386716368/ 386716367/ 386716366/ 386716365/ missing DSM 2266 uid162033 5003 5620 669 2824 1768 Halobacteroides 1069387 1096994 435853824/ 435853825/ 435853826/ 435853813/ 435853828/ missing halobius DSM 5150 5004 5621 670 2825 1769 uid184862 435853827/ 2826 Klebsiella oxytoca 604335 617733 397655648/ 397655647/ 397655646/ missing 397655645/ 397655644/ E718 uid170256 5005 5622 671 1770 4350 Lactobacillus 1026146 1040270 385817604/ 385817605/ 385817606/ 385817607/ 385817611/ missing amylovorus GRL1118 5006 5623 672 2827 1771 uid160233 Lactobacillus casei 2008783 2025228 301067096/ 301067095/ 301067094/ 301067091/ 301067090/ 301067089/ Zhang uid50673 5007 5624 673 2828 1772 4351 301067093/ 2829 Lactobacillus helveticus 1063828 1077153 385813809/ 385813808/ 385813807/ 385813806/ 385833804/ missing H10 uid162017 5008 5625 674 2830 1773 Lactobacillus helveticus 1053700 1068738 403515037/ 403515036/ 403515035/ 403515034/ 403515031/ missing R0052 uid174439 5009 5626 675 2831 1774 Lactobacillus johnsonii 939269 953168 268319510/ 268319509/ 268319508/ 268319507/ 268319503/ missing FI9785 uid41735 5010 5627 676 2832 1775 Lactobacillus reuteri 1310070 1322351 338203658/ 338203659/ 338203660/ 338203661/ 338203664/ missing SD2112 uid55357 5011 5628 677 2833 1776 Lactobacillus 2148408 2164793 385828739/ 385828738/ 385828737/ 385828734/ 385828733/ 385828732/ rhamnosus GG 5012 5629 678 2834 1777 4352 uid161983 385828736/ 2835 Lactobacillus 2154002 2170387 258509095/ 258509094/ 258509093/ 258509090/ 258509089/ 258509088/ rhamnosus GG 5013 5630 679 2836 1778 4353 uid59313 258509092/ 2837 Leuconostoc kimchii 34359 50836 296110181/ 296110180/ 296110179/ 296110176/ 296110175/ 296110174/ IMSNU 11154 5014 5631 680 2838 1779 4354 uid48589 296110178/ 2839 Methanobrevibacter 1795593 1818647 148643809/ 148643810/ 148643811/ 148643803/ 148643813/ 148643814/ smithii ATCC 35061 5015 5632 681 2840 1780 6165 uid58827 148643804/ 2841 148643805/ 2842 148643806/ 2843 148643807/ 2844 148643808/ 2845 Methanoculleus 985372 997463 397780113/ 397780112/ 397780111/ 397780110/ 397780109/ 397780108/ bourgensis MS2 5016 5633 682 2846 1781 4355 uid171377 Methanolobus 146902 163611 missing 410669356/ 410669357/ 410669358/ 410669363/ 410669364/ psychrophilus R15 5634 683 2847 1782 4356 uid177925 410669360/ 2848 Methanomethylovorans 1359134 1396372 missing 435851551/ 435851550/ 435851525/ 435851539/ 435851538/ hollandica DSM 15978 5635 684 2849 1783 4357 uid184864 435851546/ 2850 435851549/ 2851 Methanosarcina 2919153 2939722 missing 20091206/ 20091205/ 20091204/ 20091198/ 20091197/ acetivorans C2A 5636 685 2852 1784 4358 uid57879 Methanosarcina mazei 193892 214864 missing 21226255/ 21226256/ 21226258/ 21226265/ 21226266/ Go1 uid57893 5637 686 2853 1785 4359 Methanospirillum 1213815 1228530 88602375/ 88602374/ 88602373/ 88602371/ 88602368/ 88602367/ hungatei JF 1 uid58181 5017 5638 687 2854 1786 4360 Methanospirillum 2137148 2153998 missing 88603149/ 88603150/ 88603151/ 88603159/ 88603160/ hungatei JF 1 uid58181 5639 688 2855 1787 4361 Microlunatus 4106044 4121700 336119446/ 336119447/ 336119448/ 336119449/ 336139454/ 336119455/ phosphovorus NM 1 5018 5640 689 2856 1788 4362 uid68055 336119450/ 2857 336119452/ 2858 Moorella thermoacetica 2331989 2347051 83591065/ 83591064/ 83591063/ 83591059/ 83591058/ 83591057/ ATCC 39073 uid58051 5019 5641 690 2859 1789 4363 83591062/ 2860 Parvularcula 1068558 1081391 304320704/ 304320703/ 304320702/ 304320701/ 304320700/ 304320699/ bermudensis 5020 5642 691 2861 1790 4364 HTCC2503 uid51641 Pectobacterium 3681934 3716221 403059706/ 403059707/ 403059708/ 403059688/ 403059710/ 403059711/ carotovorum PCC21 5021 5643 692 2862 1791 4365 uid174335 403059709/ 2863 Pelobacter propionicus 2459983 2475529 118580706/ 118580705/ 118580704/ 118580703/ 118580699/ 118580698/ DSM 2379 uid58255 5022 5644 693 2864 1792 4366 Pelodictyon 2122302 2136560 194337066/ 194337067/ 194337068/ 194337069/ 194337071/ 194337072/ phaeoclathratiforme BU 5023 5645 694 2865 1793 4367 1 uid58173 Photorhabdus 470261 481491 253987910/ 253987911/ 253987912/ 253987913/ 253987914/ Missing asymbiotica uid59243 5024 5646 695 2866 1794 Polaromonas JS666 120587 142418 91790841/ 91790842/ 91790843/ 91790846/ 91790850/ 91790851/ uid58207 5025 5647 696 2867 1795 4368 Pseudomonas 940354 955546 330807432/ 330807433/ 330807434/ 330807435/ 330807437/ 330807438/ brassicacearum 5026 5648 697 2868 1796 4369 NFM421 uid66303 Pseudomonas stutzeri 1382383 1400530 392420331/ 392420330/ 392420329/ 392420323/ 392420322/ 392420321/ CCUG 29243 5027 5649 698 2869 1797 4370 uid168379 392420328/ 2870 Psychrobacter 1973411 1984296 93006435/ 93006434/ 93006433/ 93006432/ 93006431/ Missing cryohalolentis K5 5028 5650 699 2871 1798 uid58373 Rhodococcus 3099107 3111990 226306385/ 226306384/ 226306383/ 226306382/ 226306381/ 226306380/ erythropolis PR4 5029 5651 700 2872 1799 4371 uid59019 Rhodopseudomonas 1186657 1200150 192289534/ 192289535/ 192289536/ 192289537/ 192289538/ 192289539/ palustris TIE 1 5030 5652 701 2873 1800 4372 uid58995 Saccharophagus 3482579 3496946 90022404/ 90022403/ 90022402/ 90022401/ 90022398/ 90022397/ degradans 2 40 5031 5653 702 2874 1801 4373 uid57921 Salmonella enterica 4749510 4764672 378453463/ 378453462/ 378453461/ 378453460/ 378453457/ 378453456/ serovar Typhimurium 5032 5654 703 2875 1802 4374 14028S uid86059 Salmonella enterica 4755689 4770857 383499057/ 383499056/ 383499055/ 383499054/ 383499051/ 383499050/ serovar Typhimurium 5033 5655 704 2876 1803 4375 798 uid158047 Salmonella enterica 4736677 4751839 16767742/ 16767741/ 16767740/ 16767739/ 16767736/ 16767735/ serovar Typhimurium 5034 5656 705 2877 1804 4376 LT2 uid57799 Salmonella enterica 4757260 4772422 378702331/ 378702330/ 378702329/ 378702328/ 378702325/ 378702324/ serovar Typhimurium 5035 5657 706 2878 1805 4377 SL1344 uid86645 Salmonella enterica 4757261 4772429 379703735/ 379703734/ 379703733/ 379703732/ 379703729/ 379703728/ serovar Typhimurium 5036 5658 707 2879 1806 4378 ST4 74 uid84393 Salmonella enterica 4834059 4849227 378987165/ 378987364/ 378987163/ 378987162/ 378987159/ 378987158/ serovar Typhimurium 5037 5659 708 2880 1807 4379 T000240 uid84397 Salmonella enterica 4758667 4773829 378447805/ 378447804/ 378447803/ 378447802/ 378447799/ 378447798/ serovar Typhimurium 5038 5660 709 2881 1808 4380 uid86061 Salmonella enterica 4697115 4712277 378991759/ 378991758/ 378991757/ 378991756/ 378991753/ 378991752/ serovar Typhimurium 5039 5661 710 2882 1809 4381 UK 1 uid87049 Selenomonas sputigena 820667 833330 330838573/ 330838574/ 330838575/ 330838576/ 330838578/ Missing ATCC 35185 uid55329 5040 5662 711 2883 1810 Shewanella ANA 3 2142947 2155062 117920263/ 117920264/ 117920265/ 117920266/ 117920268/ Missing uid58347 5041 5663 712 2884 1811 Shewanella MR 4 2018034 2030135 113970045/ 113970046/ 113970047/ 113970048/ 113970050/ Missing uid58345 5042 5664 713 2885 1812 Slackia 349608 366195 257063043/ 257063042/ 257063041/ 257063038/ 257063040/ 257063036/ heliotrinireducens DSM 5043 5665 714 2886 1813 4382 20476 uid59051 257063039/ 2887 Spirosoma linguale 5893753 5908214 284039689/ 284039690/ 284039691/ 284039694/ 284039695/ 284039696/ DSM 74 uid43413 5044 5666 715 2888 1814 4383 Sulfuricurvum kujiense 1691724 1708885 313682809/ 313682810/ 313682811/ 313682812/ 313682819/ 313682820/ DSM 16994 uid60789 5045 5667 716 2889 1815 4384 313682817/ 2890 Synechococcus PCC 875746 901348 427712028/ 427712029/ 427712030/ 427712018/ 427712035/ 427712036/ 6312 uid182934 5046 5668 717 2891 1816 4385 427712032/ 2892 Syntrophomonas wolfei 1119627 1137082 114566519/ 114566520/ 114566521/ 114566522/ 114566529/ 114566530/ Goettingen uid58179 5047 5669 718 2893 1817 4386 114566525/ 2894 114566528/ 2895 Syntrophomonas wolfei 2823944 2853705 114568002/ 114568001/ 114568000/ 114567997/ 114567995/ 114567994/ Goettingen uid58179 5048 5670 719 2896 1818 4387 114567999/ #N/A 2897 114568012/ #N/A 2898 Syntrophus 932420 951906 85858754/ 85858752/ 85858751/ 85858747/ 85858744/ 85858743/ aciditrophicus SB 5049 5671 720 2899 1819 4388 uid58539 Tepidanaerobacter 565314 579879 438001500/ 438001501/ 438001502/ 438001506/ 438001508/ 438001509/ acetatoxydans Re1 5050 5672 721 2900 1820 4389 uid184827 438001507/ 2901 Tepidanaerobacter Re1 564518 579375 332798527/ 332798528/ 332798529/ 332798531/ 332798532/ 332798533/ uid66873 5051 5673 722 2902 1821 4390 Thauera MZ1T 325210 347552 217968744/ 217968745/ 217968746/ 217968749/ 217968754/ 217968755/ uid58987 5052 5674 723 2903 1822 4391 Thermacetogenium 2228448 2241200 410668523/ 410668522/ 410668521/ 410668520/ 410668519/ 410668518/ phaeum DSM 12270 5053 5675 724 2904 1823 4392 uid177811 Thermoanaerobacterium 483298 496321 304316004/ 304316005/ 304316006 304316007/ 304316008/ 304316009/ thermosaccharolyticum 5054 5676 /725 2905 1824 4393 DSM 571 uid51639 Thiocystis violascens 1605822 1624356 390949850/ 390949849/ 390949848/ 390949845/ 390949842/ Missing DSM 198 uid74025 5055 5677 726 2906 1825 Thioflavicoccus mobilis 70557 88066 431933024/ 431933023/ 431933022/ 431933018/ 431933015/ 431933014/ 8321 uid184343 5056 5678 727 2907 1826 4394 431933021/ 2908 Vibrio cholerae MJ 3021293 3036340 229608800/ 229608799/ 229608798/ 229608796/ 229608795/ 229608794/ 1236 uid59387 5057 5679 728 2909 1827 4395 Vibrio cholerae O1 110486 123671 360034501/ 360034502/ 360034503/ 360034504/ 360034505/ 360034506/ 2010EL 1786 uid78933 5058 5680 729 2910 1828 4396 Zymomonas mobilis 1590155 1602973 260753603/ 260753604/ 260753605/ 260753606/ 260753607/ 260753608/ NCIMB 11163 5059 5681 730 2911 1829 4397 uid41019 Magnetospirillum 2215526 2231935 83311137/ 83311138/ 83311139/ 83311143/ 83311145/ 83311146/ magneticum AMB 1 5060 5682 731 2912 1830 4398 uid58527 83311142/ 732 Marinobacter aquaeolei 627682 647744 120553492/ 120553493/ 120553494/ 120553498/ 120553501/ 120553502/ VT8 uid59419 5061 5683 733 2913 1831 4399 Nostoc punctiforme 6986741 7000722 186685714/ 186685707/ 186685708/ 186685709/ 186685712/ 186685713/ PCC 73102 uid57767 5062 5684 734 2914 1832 4400 Rhodobacter 628475 643049 146279749/ 146279748/ Rsph17025_3734/ 146279745/ 146279744/ 146279743/ sphaeroides ATCC 5063 5685 615 2915 1833 4401 17025 uid58451 Runella slithyformis 4922398 4938169 338213691/ 338213690/ 338213689/ 338213688/ 338213683/ 338213682/ DSM 19594 uid68317 5064 5686 735 2916 1834 4402 Acidovorax sp. NO-1 4749 20769 365096841/ 365096842/ 365096843/ 365096846/ 365096849/ 365096850/ 5336 5905 1072 3214 2158 4265 Acinetobacter 33402 52203 421624927/ 421624915/ 421624973/ 421624892/ 421624935/ 421624974/ baumannii OIFC098 5341 5911 1078 3219 2164 4271 Acinetobacter 57480 72041 427425605/ 427425618/ 427425586/ 427425611/ 427425576/ 427425548/ baumannii WC-136 5342 5912 1079 3220 2165 4272 Acinetobacter sp. P8-3- 1850 16994 358012810/ 358012809/ 358012808/ 358012807/ 358012805/ 358012804/ 8 5326 5895 1062 3204 2147 4256 Actinomyces neuii 83071 96264 405982588/ 405982587/ 405982586/ 405982584/ 405982583/ 405982582/ BVS029A5 5356 5927 1094 3235 2180 4287 405982585/ 3236 Aurantimonas 1067851 1080860 90418999/ 90418998/ 90418997/ 90418996/ 90418995/ 90418994/ manganoxydans SI85- 5319 5888 1055 3197 2140 4250 9A1 Bacillus cereus 89926 103011 206975566/ 206975742/ 206975617/ 206975739/ 206975748/ 206975706/ H3081.97 5306 5875 1041 3181 2126 4236 Bacteroides ovatus SD 22479 38083 294645431/ 294645432/ 294645433/ 294645435/ 294645438/ 294645439/ CC 2a 5364 5936 1103 3246 2189 4295 294645434/ 1104 Bacteroides sp. 2_1_7 444155 466254 255014563/ 255014564/ 255014565/ 255014566/ 255014573/ 255014574/ 5312 5881 1048 3190 2133 4243 Bacteroides sp. 15816 30563 265757021/ 265757020/ 265757019/ 265757018/ 265757016/ 265757015/ 3_1_33FAA 5329 5898 1065 3207 2150 4259 Bacteroides sp. D2 958538 975774 383114785/ 383114786/ 383114787/ 383114788/ 383114792/ 383114793/ 5327 5896 1063 3205 2148 4257 Bacteroides sp. 2_1_22 149553 165156 262405135/ 262405136/ 262405137/ 262405138/ 262405141/ 262405142/ 5346 5916 1083 3224 2169 4276 Clostridium perfringens 27368 50007 169343970/ 169343964/ 169343946/ 169343972/ 169343967/ 169343977/ C str. JGS1495 5355 5926 1093 3234 2179 4286 Collinsella aerofaciens 42484 56961 139439063/ 139439064/ 139439065/ 139439066/ 139439069/ 139439070/ ATCC 25986 5309 5878 1045 3186 2130 4240 139439068/ 3187 Coprobacillus sp. 950856 973460 365831804/ 365831805/ 365831806/ 365831813/ 365831816/ 365831817/ 3_3_56FAA 5367 5939 1109 3251 2192 4298 365831811/ 1110 Desulfonatronospira 1059989 1073987 missing 298528557/ 298528556/ 298528555/ 298528552/ 298528551/ thiodismutans ASO3-1 5906 1073 3215 2159 4266 Enterobacter 1757377 1772326 334125669/ 334125670/ 334125671/ 334125672/ 334125675/ 334125676/ hormaechei ATCC 5289 5859 1023 3159 2108 4218 49162 Enterococcus faecium 192 13547 425040991/ 425040992/ 425040993/ 425040994/ 425040995/ 425040996/ 511 5316 5885 1052 3194 2137 4247 Enterococcus faecium 141305 154660 431534711/ 431534712/ 431534713/ 431534714/ 431534715/ 431534716/ E1731 5294 5864 1028 3164 2113 4223 Enterococcus faecium 294802 308157 431753899/ 431753900/ 431753901/ 431753902/ 431753903/ 431753904/ E2883 5357 5928 1095 3237 2181 4288 Enterococcus faecium 192 13547 314941479/ 314941480/ 314941481/ 314941482/ 314941483/ 314941484/ TX0133C 5310 5879 1046 3188 2131 4241 Escherichia coli 4.0522 178149 191595 417191592/ 417191528/ 417191550/ 417191518/ 417191624/ 417191569/ 5344 5914 1081 3222 2167 4274 Escherichia coli B41 177464 190841 417293178/ 417293264/ 417292957/ 417293087/ 417293167/ 417293248/ 5358 5929 1096 3238 2182 4289 Escherichia coli B799 163075 176512 423709631/ 423709630/ 423709629/ 423709628/ 423709627/ 423709626/ 5291 5861 1025 3161 2110 4220 Escherichia coli 68462 81854 419343549/ 419343548/ 419343546/ 419343544/ 419343543/ 419343542/ DEC13A 5366 5938 1107 3249 2191 4297 419343547/ 419343545/ 1108 3250 Escherichia coli 154888 168171 419368024/ 419368147/ 419368080/ 419368110/ 419368013/ 419368125/ DEC13E 5365 5937 1105 3247 2190 4296 419368151/ 419368121/ 1106 3248 Escherichia coli 502 13913 419383946/ 419383945/ 419383944/ 419383942/ 419383941/ 419383940/ DEC14C 5299 5869 1033 3171 2118 4228 419383943/ 3172 Escherichia coli 165882 179328 419230123/ 419230122/ 419230121/ 419230120/ 419230119/ 419230118/ DEC9A 5347 5918 1085 3226 2171 4278 Escherichia coli 126845 140290 419240873/ 419240872/ 419240871/ 419240869/ 419240868/ 419240867/ DEC9C 5298 5868 1032 3169 2117 4227 419240870/ 3170 Escherichia coli 302405 315851 419246584/ 419246583/ 419246582/ 419246581/ 419246580/ 419246579/ DEC9D 5339 5909 1076 3217 2162 4269 Escherichia coli 50533 63979 419894759/ 419894758/ 419894757/ 419894756/ 419894755/ 419894754/ O111:H8 str. CVM9574 5350 5921 1088 3229 2174 4281 Escherichia coli 70169 83615 420088377/ 420088376/ 420088375/ 420088374/ 420088373/ 420088372/ O111:H8 str. CVM9602 5303 5873 1038 3178 2123 4233 Faecalibacterium 28608 45887 160944222/ 160944223/ 160944224/ 160944228/ 160944230/ 160944231/ prausnitzii M21/2 5325 5894 1061 3203 2146 4255 Fusobacterium 64292 79742 419841769/ 419841708/ 419843735/ 419841801/ 419841754/ Missing necrophorum subsp 5361 5932 1099 3241 2185 Fusobacterium sp. 435830 468937 340752713/ 340752714/ 340752704/ 340752707/ 340752711/ 340752712/ 2_1_31 5317 5886 1053 3195 2138 4248 Fusobacterium sp. 7_1 589947 618478 237744585/ 237744584/ 237744599/ 237744596/ 237744587/ 237744586/ 5354 5925 1092 3233 2178 4285 Glaciecola lipolytica E3 6001 19207 410637162/ 410637163/ 410637164/ 410637165/ 410637166/ 410637167/ 5295 5865 1029 3165 2114 4224 Lachnospiraceae 81164 93860 373469122/ 373469123/ 373469124/ 373469126/ 373469127/ Missing bacterium oral taxon 5332 5901 1068 3210 2153 082 str 373469128/ 2154 Lactobacillus casei 4097 20782 418008633/ 418008632/ 418008631/ 418008628/ 418008627/ 418008626/ UW4 5293 5863 1027 3162 2112 4222 418008630/ 3163 Lactobacillus helveticus 82009 98806 260101570/ 260101569/ 260101568/ 260101567/ 260101564/ Missing DSM 20075 5323 5892 1059 3201 2144 Lactobacillus zeae 53700 70162 366087504/ 366087503/ 366087502/ 366087499/ 366087498/ 366087497/ KCTC 3804 5363 5935 1102 3244 2188 4294 366087501/ 3245 Magnetospirillum sp. 2351 15314 458920178/ 458920177/ 458920176/ missing 458920175/ 458920174/ SO-1 5338 5908 1075 2161 4268 Magnetospirillum sp. 12059 25247 458913831/ 458913830/ 458913829/ 458913827/ 458913826/ 458913825/ SO-1 5343 5913 1080 3221 2166 4273 Marinobacter sp. 23845 43291 126665997/ 126665996/ 126665995/ 126665993/ 126665990/ 126665989/ ELB17 5290 5860 1024 3160 2109 4219 Methanoplanu 1594401 1624870 missing 374629430/ 374629429/ 374629424/ 374629423/ 374629422/ 5870 1035 3174 2120 4230 374629428/ 3175 Methanoplanu 2294064 2309549 374630020/ 374630019/ 374630018/ 374630017/ 374630014/ 374630013/ 5301 5871 1036 3176 2121 4231 Methylophaga 75838 91891 335043949/ 335043948/ 335043947/ 335043946/ 335043943/ 335043942/ aminisulfidivorans MP 5349 5920 1087 3228 2173 4280 Oribacterium sp. ACB7 598004 613860 363897445/ 363897444/ 363897443/ 363897440/ 363897438/ 363897437/ 5296 5866 1030 3166 2115 4225 363897442/ 3167 Oribacterium sp. ACB8 361812 375959 395208378/ 395208454/ 395208610/ 395208247/ 395208500/ 395208399/ 5337 5907 1074 3216 2160 4267 Photobacterium sp. 74472 92346 89073849/ 89073848/ 89073847/ 89073841/ 89073839/ 89073838/ SKA34 5352 5923 1090 3231 2176 4283 Proteus mirabilis 764883 778479 425069874/ 425069875/ 425069876/ 425069878/ 425069879/ 425069880/ WGLW6 5328 5897 1064 3206 2149 4258 Pseudomonas fragi A22 86067 103151 402700379/ 402700380/ 402700381/ 402700382/ 402700385/ 402700386/ 5322 5891 1058 3200 2143 4253 Pseudomonas 2117 19238 404399334/ 404399333/ 404399332/ 404399331/ 404399328/ 404399327/ fuscovaginae UPB0736 5331 5900 1067 3209 2152 4261 Roseburia inulinivorans 2013 15569 225374528/ 225374527/ 225374526/ 225374525/ 225374523/ 225374522/ DSM 16841 5334 5903 1070 3212 2156 4263 Salmonella enterica 1280 14693 437842753/ 437842752/ 437842751/ missing 437842750/ 437842749/ subsp. enterica serovar 5292 5862 1026 2111 4221 Enteritidi Salmonella enterica 39286 52691 167551248/ 167551249/ 167551250/ 167551251/ 205357107/ 167551253/ subsp. enterica serovar 5335 5904 1071 3213 2157 4264 Saintpau Salmonella enterica 28163 43331 427646246/ 427646245/ 427646244/ 427646243/ 427646240/ 427646239/ subsp. enterica serovar 5330 5899 1066 3208 2151 4260 Typhimuriu Salmonella enterica 72 15632 417522348/ 417522347/ 417522346/ 417522343/ 417522342/ 417522341/ subsp. enterica serovar 5307 5877 1043 3183 2128 4238 Uganda str 417522344/ 3184 Sinorhizobium meliloti 157136 171638 418398872/ 418398871/ 418398870/ 418398867/ 418398866/ 418398865/ CCNWSX0020 5320 5889 1056 3198 2141 4251 Sphingobium 37182 50024 381199745/ 381199746/ 381199747/ 381199748/ 381199749/ 381199750/ yanoikuyae XLDN2-5 5321 5890 1057 3199 2142 4252 Vibrio cholera CIRS 6477 19662 255743726/ missing 255743727/ 255743728/ 255743729/ 255743730/ 101 5304 1039 3179 2124 4234 Vibrio cholerae 131961 147079 422306078/ 422306079/ 422306080/ 422306082/ 422306083/ 422306084/ CP1035(8) 5313 5882 1049 3191 2134 4244 Vibrio cholerae HC- 4662 17826 423152696/ 423152697/ 423152698/ 423152699/ 423152700/ 423152701/ 22A1 5360 5931 1098 3240 2184 4291 Vibrio cholerae HC- 7323 20508 418336064/ 418336065/ 418336066/ 418336067/ 418336068/ 418336069/ 23A1 5359 5930 1097 3239 2183 4290 Vibrio cholerae HC- 4645 17830 418342900/ 418342901/ 418342902/ 438342903/ 418342904/ 418342905/ 28A1 5302 5872 1037 3177 2122 4232 Vibrio cholerae HC- 4645 17830 423155502/ 423155503/ 423155504/ 423155505/ 423155506/ 423155507/ 32A1 5340 5910 1077 3218 2163 4270 Vibrio cholerae HC- 594 12995 missing 422890702/ 422890703/ 422890704/ 422890705/ 422890706/ 40A1 5917 1084 3225 2170 4277 Vibrio cholerae HC- 4639 17824 424621185/ 424621186/ 424621187/ 424621188/ 424621189/ 424621190/ 47A1 5345 5915 1082 3223 2168 4275 Vibrio cholerae HC- 547 12948 missing 423164023/ 423164024/ 423164025/ 423164026/ 423164027/ 48B2 5876 1042 3182 2127 4237 Vibrio cholerae HC- 56848 70060 424015508/ 424015509/ 424015510/ 424015511/ 424015512/ 424015513/ 55B2 5348 5919 1086 3227 2172 4279 Vibrio cholerae HC- 56864 70076 423878984/ 423878985/ 423878986/ 423878987/ 423878988/ 423878989/ 60A1 5318 5887 1054 3196 2139 4249 Vibrio cholerae HC- 57077 70289 419832015/ missing 419832016/ 419832017/ 419832018/ 419832019/ 61A2 5308 1044 3185 2129 4239 Vibrio cholerae HC- 4639 17824 443514284/ 443514285/ 443514286/ 443514287/ 443514288/ 443514289/ 68A1 5324 5893 1060 3202 2145 4254 Vibrio cholerae HC- 4637 17822 443518098/ 443518099/ 443518100/ 443518101/ 443518102/ 443518103/ 71A1 5305 5874 1040 3180 2125 4235 Vibrio cholerae HC- 4635 17820 443534356/ 443534357/ 443534358/ 443534359/ 443534360/ 443534361/ 80A1 5333 5902 1069 3211 2155 4262 Vibrio cholerae HC- 4631 17816 424655756/ 424655757/ 424655758/ 424655759/ 424655760/ 424655761/ 81A2 5288 5858 1022 3158 2107 4217 Vibrio cholerae 7324 20488 417815509/ 417815510/ 417815511/ 417815512/ 417815513/ 417815514/ HCUF01 5315 5884 1051 3193 2136 4246 Vibrio cholerae MO10 324004 339140 254850926/ 254850927/ 254850928/ 254850931/ 254850932/ 254850933/ 5314 5883 1050 3192 2135 4245 Vibrio cholerae O1 str. 4654 17839 472152150/ 472152151/ 472152152/ 472152153/ 472152154/ 472152155/ EC-0012 5297 5867 1031 3168 2116 4226 Vibrio cholerae O1 str. 4648 17833 472383764/ 472183765/ 472183766/ 472183767/ 472183768/ 472183769/ EM-1546 5287 5857 1021 3157 2106 4216 Vibrio cholerae O1 str. 4648 17833 472202869/ 472202870/ 472202871/ 472202872/ 472202873/ 472202874/ NHCC-006C 5351 5922 1089 3230 2175 4282 Vibrio cholerae HC- 44947 58111 421338424/ 421338423/ 421338422/ 421338421/ 421338420/ 421338419/ 20A2 5362 5933 1100 3242 2186 4292 Vibrio cholerae HC- 42267 55452 423147143/ 423147142/ 423147141/ 423147140/ 423147139/ 423147138/ 21A1 5311 5880 1047 3189 2132 4242 Vibrio cholerae HC- 42245 55430 424618577/ 424618576/ 424618575/ 424618574/ 424618573/ 424618572/ 42A1 5353 5924 1091 3232 2177 4284 Vibrio cholerae HC- 58485 71697 424630791/ missing 424630790/ 424630789/ 424630788/ 424630787/ 51A1 5300 1034 3173 2119 4229 Xanthomonas 3685 17230 missing 325918008/ 325918007/ 325918006/ 325918004/ 325918003/ vesicatoria ATCC 5934 1101 3243 2187 4293 35937 Acetivibrio 169996 184667 366164462/ 366164461/ 366164460/ 366164459/ 366164456/ 366164455/ cellulolyticus CD2 5174 5766 893 3031 1982 4105 Acinetobacter 10653 23830 457996552/ 457996553/ 457996554/ 457996555/ 457996556/ 457996557/ baumannii AB5256 5162 5755 880 3018 1969 4092 Acinetobacter 55392 70524 417550475/ 417550474/ 417550828/ 417550745/ 417551043/ 417550931/ baumannii Naval-18 5190 5780 910 3049 1999 4120 Acinetobacter johnsonii 37452 52595 262371249/ 262371248/ 262371247/ 262371246/ 262371244/ 262371243/ SH046 5119 5720 836 2974 1925 4052 Acinetobacter junii 131814 145059 262374463/ 262374464/ 262374465/ 262374466/ 262374467/ 262374468/ SH205 5121 5722 838 2976 1927 4053 Acinetobacter lwoffii 39119 53440 262377711/ 262377712/ 262377713/ 262377714/ 262377716/ 262377717/ SH145 5181 5772 901 3039 1990 4031 262377715/ 3040 Acinetobacter 6083 19313 421857671/ 421857672/ 421857673/ 421857674/ 421857675/ 421857676/ radioresistens DSM 5141 5738 859 2996 1948 4073 6976 = NBRC 102413 Acinetobacter sp. SH024 10059 24620 293611304/ 293611305/ 293611306/ 293611307/ 293611309/ 293611310/ 5184 5775 904 3043 1993 4114 Actinomyces sp. ICM47 13893 28963 396584771/ 396584768/ 396584764/ 396584767/ 396584777/ 396584762/ 5102 5703 818 2954 6204 4036 Actinomyces sp. oral 1582915 1598023 320095079/ 320095080/ 320095081/ 320095082/ 320095085/ 320095086/ taxon 178 str 5139 5735 857 2994 1946 4072 Alcanivorax pacificus 73364 89677 407801754/ 407801755/ 407801756/ 407801760/ 407801761/ 407801762/ W11-5 5128 5727 845 2982 1934 4060 Alcanivorax sp. DG881 3121028 3136375 254429639/ 254427953/ 254428479/ 254429762/ 254428445/ 254426952/ 5165 5758 883 3022 1972 4095 Bacillus cereus 790081 800966 423480947/ 423480948/ 423480949/ 423480950/ 423480951/ Missing BAG6X1-2 5252 5831 976 3116 2065 Bacillus cereus HuB2-9 3250049 3263169 423535446/ 423535445/ 423535444/ 423535443/ 423535442/ 423535441/ 5246 5826 969 3108 2058 4173 Bacteroides coprophilus 12964 26509 224023324/ 224023323/ 224023322/ 224023320/ 224023319/ 224023318/ DSM 18228 5281 5700 1009 6194 2098 4209 Bacteroides ovatus 2284234 2301803 423288076/ 423288075/ 423288074/ 423288073/ 423288070/ 423288069/ CL02T12C04 5150 5744 868 3005 1957 4081 Bacteroides ovatus SD 59559 81656 293373691/ 293373690/ 293373689/ 293373688/ 293373679/ 293373678/ CMC 3f 5144 5740 862 2999 1951 4076 Bacteroides sp. 1_1_14 157533 186706 298384296/ 298384295/ 298384294/ missing 298384292/ 298384291/ 5282 5851 1011 2099 4210 Bacteroides sp. D1 413277 428835 345508162/ 237715294/ 345508160/ 237715296/ 345508156/ 345508155/ 5278 5699 1006 6196 2095 4207 Bacteroides 90276 105878 294807073/ 294807072/ 294807071/ 294807069/ 294807066/ 294807065/ xylanisolvens SD CC 5276 5698 1003 6190 2092 4204 1b Bifidobacterium 878442 897214 229817499/ 229817500/ 229817501/ ZP_03449450/ 229817507/ 229817508/ angulatum DSM 20098 5126 5726 843 6192 1932 4058 Bifidobacterium 7024 23499 421735410/ 421735409/ 421735408/ 421735407/ 421735404/ 421735403/ bifidum IPL 5143 5739 861 2998 1950 4075 Bifidobacterium 939991 958805 313140117/ 313140118/ 313140119/ 313140120/ 313140123/ 313140124/ bifidum NCIMB 41171 5169 5760 887 3026 1976 4099 Brachybacterium 53546 66327 386775206/ 386775207/ 386773208/ 386775209/ 386775210/ 386775211/ paraconglomeratum 5214 5799 936 3076 2025 4143 LC44 Brevibacterium 42197 55309 295396113/ 295396312/ 295396111/ 295396110/ 295396108/ 295396107/ mcbrellneri ATCC 5199 5789 920 3060 2009 4129 49030 Cellvibrio sp. BR 223559 242477 388258095/ 388258094/ 388258093/ 388258089/ 388258086/ 388258085/ 5191 5781 911 3050 2000 4121 388258091/ 3051 Clostridium butyricum 1158571 1173561 237668144/ 237668630/ 237667881/ 237668455/ 237668352/ 237667216/ E4 str. BoNT E BL5262 5189 5779 909 3048 1998 4119 Clostridium perfringens 53370 80361 182624257/ 182624189/ 182624228/ 182624175/ 182624223/ 182624227/ D str. JGS1721 5218 5803 940 3080 2029 4146 Collinsella intestinalis 30782 46221 229814947/ 229814946/ 229814945/ 229814944/ 229814938/ 229814937/ DSM 13280 5157 5750 875 3013 1964 4087 Collinsella stercoris 31011 46881 210635259/ 210635260/ 210635261/ 210635262/ 210635282/ 210635283/ DSM 13279 5235 5818 958 3097 2047 4163 Coprobacillus sp. 100734 114652 374625977/ 374625978/ 374625979/ 374625981/ 374625983/ 374625984/ 8_2_54BFAA 5107 5708 823 2959 1911 4040 Cylindrospermopsis 2528 15922 missing 282900779/ 282900778/ 282900774/ 282900772/ 282900771/ raciborskii CS-505 5855 1019 6198 2104 4215 delta proteobacterium 35975 51793 301064129/ truncated 301064167/ 301064133/ 301064130/ 301064155/ NaphS2 5195 915 3055 2004 4125 Desulfotomaculum 100510 112411 357040623/ 357040624/ 357040625/ 357040626/ 357040629/ Missing gibsoniae DS 5211 5796 932 3072 2021 Enterobacter sp. SST3 61939 75308 401676813/ 401676814/ 401676815/ 401676816/ 401676817/ 401676818/ 5243 5824 966 3105 2055 4170 Enterococcus faecalis 21896 38028 307287298/ 307287299/ 307287300/ 307287301/ 307287305/ 307287306/ TX0109 5132 5730 850 2987 1939 4065 Enterococcus faecalis 21900 38032 422702471/ 422702472/ 422702473/ 422702474/ 422702478/ 422702479/ TX1302 5237 5820 960 3099 2049 4164 Enterococcus faecium 9609 22964 425047892/ 425047891/ 425047890/ 425047889/ 425047888/ 425047887/ 509 5407 5797 934 3074 2023 4141 Enterococcus faecium 192 13547 425034027/ 425034028/ 425034029/ 425034030/ 425034031/ 425034032/ 514 5164 5757 882 3021 1971 4094 Enterococcus faecium 5282 18637 425024182/ 425024181/ 425024180/ 425024179/ 425024178/ 425024177/ C1904 5405 5785 916 3056 2005 4126 Enterococcus faecium 142 13497 425019537/ 425019538/ 425019539/ 425019540/ 425019541/ 425019542/ C497 5151 5745 869 3006 1958 4030 Enterococcus faecium 308557 323090 430834457/ 430834456/ 430834455/ 430834454/ 430834452/ 430834451/ E0679 5187 5777 907 3046 1996 4117 Enterococcus faecium 314134 328775 431640912/ 431640911/ 431640910/ 431640909/ 431639958/ 431639957/ E1904 5265 5841 990 3133 2079 4191 Enterococcus faecium 5283 18638 424979863/ 424979862/ 424979861/ 424979860/ 424979859/ 424979858/ ERV99 5399 5709 824 2960 1912 4041 Enterococcus faecium 191 13546 424977969/ 424977970/ 424977971/ 424977972/ 424977973/ 424977974/ P1123 5203 5792 924 3064 2013 4132 Enterococcus faecium 5282 18637 424974922/ 424974921/ 424974920/ 424974919/ 424974918/ 424974917/ P1137 5414 5848 1001 3144 2090 4202 Enterococcus faecium 191 13546 424971162/ 424971163/ 424971164/ 424971165/ 424971166/ 424971167/ P1139 5204 5793 925 3065 2014 4133 Enterococcus faecium 9607 22962 314950597/ 314950596/ 314950595/ 314950594/ 314950593/ 314950592/ TX0133A 5404 5768 895 3033 1984 4106 Enterococcus faecium 192 13547 314996683/ 314996684/ 314996685/ 314996686/ 314996687/ 314996688/ TX0133a01 5244 5825 967 3106 2056 4171 Enterococcus faecium 9609 22964 314939181/ 314939180/ 314939179/ 314939178/ 314939177/ 314939176/ TX0133a04 5409 5827 970 3109 2059 4174 Enterococcus faecium 9609 22964 314992451/ 314992450/ 314992449/ 314992448/ 314992447/ 314992446/ TX0133B 5408 5804 941 3081 2030 4147 Escherichia coli 2534- 70489 83934 417589503/ 417589502/ 417589501/ 417589499/ 417589498/ 417589497/ 86 5275 5849 1002 3145 2091 4203 417589500/ 3146 Escherichia coli 3.3884 1217008 1230445 417270173/ 417270040/ 417269258/ 417268711/ 417269719/ 417270270/ 5116 5717 833 2970 1922 4049 Escherichia coli 96.154 246141 259589 417223908/ 417224699/ 417224010/ 417225276/ 417224519/ 417225329/ 5285 5854 1016 3155 2102 4213 417225258/ 1017 Escherichia coli 318305 331717 419281130/ 419281129/ 419281128 419281126/ 419281125/ 419281124/ DEC10E 5118 5719 2972 1924 4051 419281127/ 2973 Escherichia coli 147401 160847 419281503/ 419281502/ 419281501 419281500/ 419281499/ 419281498/ DEC10F 5225 5809 3088 2037 4154 Escherichia coli 92227 105618 419352610/ 419352609/ 419352607/ 419352604/ 419352603/ 419352602/ DEC13B 5284 5853 1014 3153 2101 4212 419352608/ 419352606/ 1015 3154 Escherichia coli 68089 81481 419358139/ 419358138/ 419358136/ 419358134/ 419358133/ 419358132/ DEC13C 5283 5852 1012 3151 2100 4211 419358137/ 419358135/ 1013 3152 Escherichia coli 138809 154330 419363104/ 419363100/ 419363098/ 419363096/ 419363095/ 419363094/ DEC13D 5277 5850 1004 3147 2093 4205 419363099/ 419363097/ 1005 3148 Escherichia coli 143274 156684 419378584/ 419378583/ 419378582/ 419378580/ 419378578/ 419378576/ DEC14B 5250 5829 974 3113 2063 4178 419378581/ 3114 Escherichia coli 9353 22765 419389188/ 419389187/ 419389186/ 419389184/ 419389183/ 419389182/ DEC14D 5156 5749 874 3011 1963 4086 419389185/ 3012 Escherichia coli 11328 24717 419173689/ 419173698/ 419173682/ 419173671/ 419173696/ 419173675/ DEC7B 5109 5710 826 2962 1914 4043 Escherichia coli 78187 91632 419200151/ 419200150/ 419200149/ 419200148/ 419200147/ 419200146/ DEC8A 5200 5790 921 3061 2010 4130 Escherichia coli 18788 33547 419206519/ 419206636/ 419206641/ 419206658/ 419206669/ 419206621/ DEC8B 5227 5811 950 3090 2039 4156 419206666/ 5812 Escherichia coli 139979 153424 419235451/ 419235547/ 419235503/ 419235607/ 419235606/ 419235530/ DEC9B 5124 5724 841 2979 1930 4056 419235615/ 2980 Escherichia coli DEC9E 26957 40402 419252319/ 419252318/ 419252317/ 419252315/ 419252314/ 419252313/ 5261 5838 985 3127 2074 4187 419252316/ 3128 Escherichia coli JB1-95 70264 85023 417209283/ 417209341/ 417209350/ 417209346/ 417209331/ 417209313/ 5267 5842 992 3135 2081 4193 Escherichia coli KD2 14059 28959 419919262/ 419919261/ 419919260/ 419919259/ 419919256/ 419919255/ 5403 5761 888 3027 1977 4100 Escherichia coli KTE12 333731 347168 432379522/ 432379521/ 432379520/ 432379519/ 432379518/ 432379517/ 5106 5707 822 2958 1910 4039 Escherichia coli 791 14168 433099196/ 433099195/ 433099194/ 433099193/ 433099192/ 433099191/ KTE139 5154 5748 872 3009 1961 4084 Escherichia coli 3821 17182 433318462/ 433118461/ 433118460/ 433118459/ 433118458/ 433118457/ KTE153 5401 5729 849 2986 1938 4064 Escherichia coli 421523 434900 432983125/ 432983126/ 432983127/ 432983128/ 432983129/ 432983130/ KTE211 5161 5754 879 3017 1968 4091 Escherichia coli 593208 606594 432993615/ 432993616/ 432993617/ 432993618/ 432993619/ 432993620/ KTE218 5198 5788 919 3059 2008 4128 Escherichia coli 445622 459002 432531967/ 432531966/ 432531965/ 432531964/ 432531963/ 432531962/ KTE234 5239 5822 962 3101 2051 4166 Escherichia coli 290106 305021 432551900/ 432551901/ 432551902/ 432551903/ 432551906/ 432551907/ KTE47 5233 5816 956 3095 2045 4161 Escherichia coli 325634 339020 432566734/ 432566735/ 432566736/ 432566737/ 432566738/ 432566739/ KTE53 5264 5840 989 3132 2078 4190 Escherichia coli 789918 803295 432708021/ 432708022/ 432708023/ 432708024/ 432708025/ 432708026/ KTE6 5183 5774 903 3042 1992 4113 Escherichia coli MS 69- 8280 23192 301024309/ 301024308/ 301024307/ 301024306/ 301024303/ 301024302/ 1 5147 5742 865 3002 1954 4079 Escherichia coli 1722 15111 442594978/ 442594979/ 442594980/ 442594981/ 442594982/ 442594983/ O10:K5(L):H4 str. 5159 5752 877 3015 1966 4089 ATCC 23506 Escherichia coli 43889 57335 419887469/ 419887468/ 419887467/ 419887466/ 419887465/ 419887464/ O111:H8 str. CVM9570 5220 5806 943 3083 2032 4149 Escherichia coli 13485 26931 420093467/ 420093468/ 420093469/ 420093470/ 420093471/ 420093472/ O111:H8 str. CVM9634 5134 5732 852 2989 1941 4067 Escherichia coli 111648 125096 458688575/ 458688574/ 458688573/ 458688572/ 458688571/ 458688570/ O113:H21 str. CL-3 5271 5844 996 3139 2085 4197 Escherichia coli 179 13555 458890352/ 458890353/ 458890354/ 483041219/ 458890355/ 458890356/ O25b:ST131 str. JIE186 5163 5756 881 3019 1970 4093 481041220/ 3020 Escherichia coli 263790 277236 415823690/ 415823689/ 415823688/ 415823687/ 415823686/ 415823685/ OK1180 5194 5784 914 3054 2003 4124 Escherichia coli S17 4631 19699 450256213/ 450256210/ 450256209/ 450256207/ 450256206/ 450256205/ 5168 5759 886 3025 1975 4098 Escherichia coli 133003 146449 417605265/ 417605264/ 417605263/ 417605261/ 417605260/ 417605259/ STEC_94C 5113 5714 830 2966 1919 4046 417605262/ 2967 Escherichia coli 209301 222747 458059912/ 458059911/ 458059910/ 458059909/ 458059908/ 458059907/ TW10828 5138 5734 856 2993 1945 4071 Eubacterium 2986470 3002126 389577853/ 389577852/ 389577851/ 389577850/ 389577845/ Missing cellulosolvens 6 5216 5801 938 3078 2027 Faecalibacterium 463318 476823 257438028/ 257438027/ 257438026/ 257438025/ 257438023/ 257438022/ prausnitzii A2-165 5192 5782 912 3052 2001 4122 Fulvimarina pelagi 98942 111984 114706333/ 114706332/ 114706331/ 114706330/ 114706329/ 114706328/ HTCC2506 5193 5783 913 3053 2002 4123 Fusobacterium 564908 579712 373113611/ 373113608/ 373113625/ 373113618/ 373113613/ Missing necrophorum subsp 5140 5736 858 2995 1947 373113609/ 5737 Fusobacterium 33577 58056 296328615/ 296328614/ 296328627/ 296328621/ 296328617/ 296328616/ nucleatum subsp. 5179 5770 899 3037 1988 4110 nucleatum ATCC 23726 Fusobacterium sp. 557323 577812 336418217/ 336418218/ 336418208/ 336418211/ 336418215/ 336418216/ 11_3_2 5188 5778 908 3047 1997 4118 Fusobacterium sp. 1001099 1027629 294785575/ 294785574/ 294785588/ 294785585/ 294785577/ 294785576/ 3_1_27 5180 5771 900 3038 1989 4111 Fusobacterium sp. 242664 257240 317058273/ 317058274/ 317058268/ 317058270/ 317058272/ Missing 3_1_5R 5175 5767 894 3032 1983 Fusobacterium ulcerans 320598 335972 404368581/ 404368582/ 404368577/ 404368578/ 404368579/ 404368580/ ATCC 49185 5223 5807 946 3086 2035 4152 gamma proteobacterium 205372 220880 386288256/ 386288257/ 386288258/ 386288262/ 386288263/ 386288264/ BDW918 5172 5764 891 3030 1980 4103 gamma proteobacterium 77590 90394 329896084/ 329896083/ 329896082/ 329896081/ 329896080/ 329896079/ IMCC3088 5131 5728 848 2985 1937 4063 Geobacillus sp. 128797 142893 196250359/ 196250358/ 196250357/ 196250356/ 196250354/ Missing G11MC16 5232 5815 955 3094 2044 Geobacillus 1816670 1827544 423719991/ 423719992/ 423719993/ 423719994/ 423719995/ Missing thermoglucosidan 5201 5791 922 3062 2011 Glaciecola polaris LMG 41332 52442 410618148/ 196250358/ 410618146/ 410618145/ 410618144/ Missing 21857 5280 5815 1008 3150 2097 Glaciecola punicea 141624 156305 381395614/ 381395615/ 381395616/ 381395619/ 381395620/ 381395621/ DSM 14233 = ACAM 5236 5819 959 3098 2048 4033 611 Haloarcula japonica 437852 452733 448689859/ 448689858/ 448689857/ 448689855/ 448689853/ 448689852/ DSM 6131 5254 5833 978 3119 2067 4180 Halomonas sp. HAL1 3095 24448 352101114/ 352101113/ 352101112/ 5′ of gene 352101104/ 352101103/ 5125 5725 842 missing 1931 4057 Holdemania filiformis 5004 17955 223986440/ 223986439/ 223986438/ 223986437/ 223986436/ 223986435/ DSM 12042 5186 5776 906 3045 1995 4116 Holophaga foetida 8075 23271 373485785/ 373485786/ 373485787/ 373485789/ 373485791/ 373485792/ DSM 6591 5115 5716 832 2969 1921 4048 Johnsonella ignava 86362 100649 358066716/ 358066715/ 358066714/ 358066713/ 358066709/ 358066708/ ATCC 51276 5240 5823 963 3102 2052 4167 Lachnoanaerobaculum 1084944 1098333 315651036/ 315651037/ 315651038/ 315651039/ 315651043/ 315651044/ (Eubacterium) 5406 5944 1172 3278 2248 4024 saburreum DSM 3986 Lachnoanaerobaculum 2724 17869 419720906/ 419720918/ 419720908/ 419720916/ 419720919/ 419720903/ (Eubacterium) 5400 5944 1170 3276 2248 4020 saburreum F0468 Lachnospiraceae 88155 102869 336440367/ 336440366/ 336440365/ 336440363/ 336440358/ Missing bacterium 1_1_57FAA 5105 5706 821 2957 1909 Lachnospiraceae 229861 244575 317501016/ 317501017/ 317501018/ 317501020/ 317501025/ Missing bacterium 8_1_57FAA 5148 5743 866 3003 1955 Lachnospiraceae oral 111528 132974 331001729/ 331001730/ 331001731/ 331001734/ 331001737/ Missing taxon 107 str 5110 5711 827 2963 1915 331001745/ 1916 Lactobacillus casei Lpc-37 1267 17585 418013531/ 418013530/ 418013529/ 418013528/ 418013525/ 418013524/ 5182 5773 902 3041 1991 4112 Lactobacillus 142681 159146 421768513/ 421768512/ 421768511/ 421768508/ 421768507/ 421768506/ rhamnosus LRHMDP2 5255 5834 979 3120 2068 4181 421768510/ 3121 Lactobacillus 129821 146256 421772571/ 421772572/ 421772573/ 421772574/ 421772577/ 421772578/ rhamnosus LRHMDP3 5253 5832 977 3117 2066 4035 421772576/ 3118 Lactobacillus johnsonii 547785 564068 227889974/ 227889975/ 227889976/ 227889977/ 227889982/ Missing ATCC 33200 5196 5786 917 3057 2006 Lactobacillus reuteri 141036 153317 227544819/ 227544818/ 227544817/ 227544816/ 227544813/ Missing CF48-3A 5411 5839 986 3129 2075 Microcystis aeruginosa 76626 89380 missing 425444979/ 425444980/ 425444983/ 425444986/ Missing PCC 9443 5856 1020 3156 2105 Nitratireductor indicus 129343 142197 407974990/ 407974989/ 407974988/ 407974987/ 407974986/ 407974985/ C115 5160 5753 878 3016 1967 4090 Opitutacea 241740 261922 373849354/ missing 373849352/ missing 373849343/ 373849342/ 5279 1007 2096 4208 Oribacterium sp. ACB1 648540 662687 363899397/ 363899396/ 363899395/ 363899394/ 363899392/ 363899391/ 5117 5718 834 2971 1923 4050 Paenibacillus elgii B69 3761 16828 357010164/ 357010163/ 357010162/ 357010161/ 357010160/ 357010159/ 5226 5810 949 3089 2038 4155 Pantoea sp. GM01 181180 196279 398800469/ 398800470/ 398800471/ 398800472/ 398800475/ 398800476/ 5153 5747 871 3008 1960 4083 Parabacteroides sp. D25 349829 371928 410103031/ 410103030/ 410103029/ 410103028/ 410103021/ 410103020/ 5146 5741 864 3001 1953 4078 Pectobacterium 12026 27179 421082308/ 421082307/ 421082306/ 421082305/ 421082302/ 421082301/ wasabiae CFBP 3304 5112 5713 829 2965 1918 4045 Pseudanabaena biceps 21843 38743 443475022/ 443475021/ 443475020/ 443475017/ 443475015/ 443475014/ PCC 7429 5135 5733 853 2990 1942 4068 Pseudoalteromona 80528 95485 409203052/ 409203051/ 409203050/ 409203049/ 409203047/ 409203046/ 5238 5821 961 3100 2050 4165 Pseudoalteromonas 7985 26765 442610065/ 442610064/ 442610063/ 442610062/ 442610058/ 442610057/ luteoviolacea B = 5217 5802 939 3079 2028 4145 ATCC 29581 Pseudomonas mandelii 1337696 1354409 407366557/ 407366556/ 407366555/ missing 407366548/ 407366547/ JR-1 5228 5813 951 2040 4157 Pseudomonas 141817 157158 359780192/ 359780191/ 359780190/ missed by 359780188/ 359780187/ psychrotolerans L19 5173 5765 892 ORF finder 1981 4104 tool Pseudomonas syringae 83306 107608 470894104/ 470894103/ 470894102/ 470894100/ 470894093/ 470894092/ Lz4W 5133 5731 851 2988 1940 4066 Roseobacter sp. 2039740 2052609 86139888/ 86139887/ 86139886/ 86139885/ 86139884/ 86139883/ MED 193 5224 5808 947 3087 2036 4153 Ruminococcaceae 39789 54066 332655416/ 332655415/ 332655414/ 332655413/ 332655411/ 332655410/ bacterium D16 5114 5715 831 2968 1920 4047 Salmonella enterica 4758712 4773847 375004441/ 375004440/ 375004439/ 375004438/ 375004435/ 375004434/ subsp. enterica serovar 5111 5712 828 2964 1917 4044 Infanti Salmonella enterica 116457 131625 422028739/ 422028738/ 422028737/ 422028736/ 422028733/ 422028732/ serovar Typhimurium 5158 5751 876 3014 1965 4088 STm1 uid181283 Salmonella enterica 348410 363578 458765354/ 458765353/ 458765352/ 458765351/ 458765348/ 458765347/ serovar Typhimurium 5170 5762 889 3028 1978 4101 ST1660 06 uid190371 Salmonella enterica 28160 43328 422033790/ 422033789/ 422033788/ 422033787/ 422033784/ 422033783/ serovar Typhimurium 5177 5769 897 3035 1986 4108 STm2 uid181284 Salmonella enterica 30253 45421 427597642/ 427597641/ 427597640/ 427597639/ 427597636/ 427597635/ serovar Typhimurium 5215 5800 937 3077 2026 4144 STm3 uid181357 Salmonella enterica 28155 43323 427557943/ 427557942/ 427557941/ 427557940/ 427557937/ 427557936/ serovar Typhimurium 5234 5817 957 3096 2046 4162 STm8 uid181355 Salmonella enterica 30250 45412 427682142/ 427682141/ 427682140/ 427682139/ 427682136/ 427682135/ serovar Typhimurium 5251 5830 975 3115 2064 4179 STm12 uid181362 Salmonella enterica 28233 43401 427622026/ 427622025/ 427622024/ 427622023/ 427622020/ 427622019/ serovar Typhimurium 5256 5835 980 3122 2069 4182 STm4 uid181358 Salmonella enterica 28172 43334 427576011/ 427576010/ 427576009/ 427576008/ 427576005/ 427576004/ serovar Typhimurium 5260 5837 984 3126 2073 4186 STm9 uid181356 Salmonella enterica 28220 43388 427658747/ 427658746/ 427658745/ 427658744/ 427658741/ 427658740/ serovar Typhimurium 5274 5847 1000 3143 2089 4201 STm11 uid181361 Salmonella enterica 28164 42133 missing missing 427664029/ 427664028/ 427664025/ 427664024/ serovar Typhimurium 1175 3149 2094 4206 STm11 uid181361 Salmonella enterica 105150 120318 167991286/ 167991285/ 167991284/ 167991283/ 167991280 167991279/ subsp. enterica serovar 5247 5828 971 3110 4175 4,[5],12:i:-str. CVM23701 Selenomonas sputigena 547454 560116 260887941/ 260887940/ 260887939/ 260887938/ 260887934/ truncated ATCC 35185 5120 5721 837 2975 1926 Shewanella baltica 211735 229091 418024977/ 418024978/ 418024979/ 418024983/ 418024984/ 418024985/ OS625 5122 5723 839 2977 1928 4054 Sphingobium indicum 80766 95394 390169179/ 390169178/ 390169177/ 390169174/ 390169173/ 390169172/ B90A 5268 5843 993 3136 2082 4194 Sporolactobacillus 5785 18771 404330921/ 404330920/ 404330919/ 404330918/ 404330917/ 404330916/ vineae DSM 21990 = 5413 5845 997 3140 2086 4198 SL153 Sporosarcina 827385 843078 340357063/ 340357064/ 340357065/ 340357066/ 340357070/ 340357071/ newyorkensis 2681 5206 5795 927 3067 2016 4135 Stomatobaculum 64338 77737 373106083/ 373106084/ 373106085/ 373106086/ 373106087/ 373106088/ longum 5410 5946 1173 3279 1905 4034 (Lachnospiraceae bacterium ACC2) Stomatobaculum 21037 34636 373106631/ 373106630/ 373106629/ 373106628/ 373106623/ Missing longum 5412 5947 1174 3280 1906 (Lachnospiraceae bacterium ACC2) Streptomyces sp. 3417840 3430694 302520235/ 302520234/ 302520233/ 302520232/ 302520231/ 302520230/ SPB78 5273 5846 999 3142 2088 4200 Thiorhodovibrio sp. 970 1742073 1757839 381159244/ Thi970DRAFT_(—) 381159246/ Thi970DRAFT_(—) 381159257/ 381159258/ 5286 2972/5701 1018 2976/6182 2103 4214 Thiothrix nivea DSM 4299231 4317524 386818309/ 386818310/ 386818311/ 386818316/ 386818318/ 386818319/ 5205 5197 5787 918 3058 2007 4127 Vibrio cholerae 4260B 149276 164412 440708890/ 440708891/ 440708892/ 440708895/ 440708896/ 440708897/ 5229 5814 952 3091 2041 4158 Vibrio cholerae B33 48656 63703 229509128/ 229509127/ 229509126/ 229509124/ 229509123/ 229509122/ 5205 5794 926 3066 2015 4134 Vibrio cholerae B33 82818 97865 153821444/ 153821461/ 153821425/ 153821428/ 153821427/ 153821414/ 5219 5805 942 3082 2031 4148 Vibrio cholerae 7472 20636 421334488/ missing 421334490/ 421334491/ 421334492/ 421334493/ CP1048(21) 5245 968 3107 2057 4172 Vibrio cholerae 4634 17819 424605792/ missing 424605794/ 424605795/ 424605796/ 424605797/ CP1050(23) 5210 931 3071 2020 4139 Vibrio cholerae H1 310730 323915 457927364/ missing 457927366/ 457927367/ 457927368/ 457927369/ 5155 873 3010 1962 4085 Vibrio cholerae HC- 4700 17885 423730156/ missing 423730158/ 423730159/ 423730160/ 423730161/ 17A1 5136 854 2991 1943 4069 Vibrio cholerae HC- 4629 17814 424001206/ missing 424001208/ 424001209/ 424001210/ 424001211/ 17A2 5145 863 3000 1952 4077 Vibrio cholerae HC- 4649 17834 423144201/ missing 423144203/ 423144204/ 423144205/ 423144206/ 19A1 5185 905 3044 1994 4115 Vibrio cholerae HC- 4631 17816 424005362/ missing 424005364/ 424005365/ 424005366/ 424005367/ 37A1 5166 884 3023 1973 4096 Vibrio cholerae HC- 4635 17820 422924882/ missing 422924884/ 422924885/ 422924886/ 422924887/ 38A1 5248 972 3111 2061 4176 Vibrio cholerae HC- 4623 17808 424609627/ missing 424609629/ 424609630/ 424609631/ 424609632/ 39A1 5263 988 3131 2077 4189 Vibrio cholerae HC- 4650 17814 424612431/ missing 424612433/ 424612434/ 424612435/ 424612436/ 41A1 5208 929 3069 2018 4137 Vibrio cholerae HC- 4886 18071 418348066/ missing 418348068/ 418348069/ 418348070/ 418348071/ 43A1 5259 983 3125 2072 4185 Vibrio cholerae HC- 7242 20406 421345611/ missing 421345612/ 421345553/ 421345630/ 421345651/ 46A1 5241 964 3103 2053 4168 Vibrio cholerae HC- 298 12976 422901578/ missing 422901580/ 422901581/ 422901582/ 422901583/ 48A1 5222 945 3085 2034 4151 Vibrio cholerae HC- 7286 20471 417812640/ missing 417812642/ 417812643/ 417812644/ 417812645/ 49A2 5242 965 3104 2054 4169 Vibrio cholerae HC- 4633 17818 424644165/ missing 424644167/ 424644168/ 424644169/ 424644170/ 56A2 5270 995 3138 2084 4196 Vibrio cholerae HC- 4631 17816 424651808/ missing 424651810/ 424651811/ 424651812/ 424651813/ 57A2 5230 953 3092 2042 4159 Vibrio cholerae HC- 6230 19394 418354574/ missing 418354555/ 418354534/ 418354592/ 418354501/ 61A1 5167 885 3024 1974 4097 Vibrio cholerae HC- 4643 17828 423891874/ missing 423891876/ 423891877/ 423891878/ 423891879/ 62A1 5249 973 3112 2062 4177 Vibrio cholerae HC- 4643 17828 424023371/ missing 424023373/ 424023374/ 424023375/ 424023376/ 62B1 5207 928 3068 2017 4136 Vibrio cholerae HC- 4641 17826 443502702/ missing 443502704/ 443502705/ 443502706/ 443502707/ 64A1 5269 994 3137 2083 4195 Vibrio cholerae HC- 4643 17828 443506617/ missing 443506619/ 443506620/ 443506621/ 443506622/ 65A1 5262 987 3130 2076 4188 Vibrio cholerae HC- 4639 17824 443510722/ missing 443510724/ 443510725/ 443510726/ 443510727/ 67A1 5127 844 2981 1933 4059 Vibrio cholerae HC- 4637 17822 424026175/ missing 424026177/ 424026178/ 424026179/ 424026180/ 69A1 5129 846 2983 1935 4061 Vibrio cholerae HC- 4639 17824 422905802/ missing 422905804/ 422905805/ 422905806/ 422905807/ 70A1 5142 860 2997 1949 4074 Vibrio cholerae HC- 4639 17824 443522965/ missing 443522967/ 443522968/ 443522969/ 443522970/ 72A2 5212 933 3073 2022 4140 Vibrio cholerae HC- 4635 17820 423926648/ missing 423926650/ 423926651/ 423926652/ 423926653/ 77A1 5202 923 3063 2012 4131 Vibrio cholerae HC- 312355 325519 443530594/ missing 443530596/ 443530597/ 443530598/ 443530599/ 7A1 5209 930 3070 2019 4138 Vibrio cholerae HC- 7298 20483 443537943/ missing 443537945/ 443537946/ 443537947/ 443537948/ 81A1 5130 847 2984 1936 4062 Vibrio cholerae HFU- 4640 17825 422912398/ missing 422912400/ 422912401/ 422932402/ 422912403/ 02 5178 898 3036 1987 4109 Vibrio cholerae O1 str. 4571 17756 458013689/ missing 458013691/ 458013692/ 458013693/ 458013694/ 2010EL-1792 5176 896 3034 1985 4107 Vibrio cholerae O1 str. 116442 118493 458067976/ 458067975/ 458067974/ 458067973/ 458067972/ 458067971/ 3582-05 5402 6206 1171 3277 2249 4029 Vibrio cholerae O1 str. 4667 17831 472149945/ missing 472149947/ 472149948/ 472149949/ 472149950/ EC-0009 5266 991 3134 2080 4192 Vibrio cholerae O1 str. 4644 17829 472157764/ missing 472157766/ 472157767/ 472157768/ 472157769/ EC-0027 5272 998 3141 2087 4199 Vibrio cholerae O1 str. 4648 17833 472166840/ missing 472166842/ 472166843/ 472166844/ 472166845/ EDC-020 5258 982 3124 2071 4184 Vibrio cholerae O1 str. 119978 135025 449054089/ 449054088/ 449054087/ 449054085/ 449054084/ 449054083/ Inaba G4222 5171 5763 890 3029 1979 4102 Vibrio cholerae O1 str. 4644 17829 472214605/ missing 472214607/ 472214608/ 472214609/ 472214610/ Nep-21106 5221 944 3084 2033 4150 Vibrio cholerae Ol str. 4644 17829 472217724/ missing 472217726/ 472217727/ 472217728/ 472217729/ Nep-21113 5149 867 3004 1956 4080 Vibrio cholerae O1 str. 4650 17835 472199358/ missing 472199360/ 472199361/ 472199362/ 472199363/ NHCC-004A 5123 840 2978 1929 4055 Vibrio cholerae O1 str. 4648 17833 472210699/ missing 472210701/ 472210702/ 472210703/ 472210704/ NHCC-010F 5231 954 3093 2043 4160 Vibrio cholerae O1 str. 4652 17837 472222753/ missing 472222755/ 472222756/ 472222757/ 472222758/ PCS-023 5108 825 2961 1913 4042 Vibrio cholerae 4335 19471 458152045/ 458152046/ 458152047/ 458152050/ 458152051/ 458152052/ VC4370 5103 5704 819 2955 1907 4037 Vibrio harveyi CAIM 2186 21054 472443545/ 472443546/ 472443547/ 472443548/ 472443552/ 472443553/ 1792 5104 5705 820 2956 1908 4038 Vibrio shilonii AK1 42036 59655 149189598/ 149189597/ 149189596/ 149189595/ 149189592/ 149189591/ 5213 5798 935 3075 2024 4142 Vibrio tubiashii ATCC 78266 91498 343503397/ 343503398/ 343503399/ 343503400/ 343503401/ 343503402/ 19109 5257 5836 981 3123 2070 4183 Vibrio cholerae O1 str. 116441 129626 458790585/ missing 458790583/ 458790582/ 458790581/ 458790580/ 2010EL-1798 5137 855 2992 1944 4070 Yersinia ruckeri ATCC 13923 29006 238756189/ 238756190/ 238756191/ 238756192/ 238756193/ 238756194/ 29473 5152 5746 870 3007 1959 4082 Thioalkalivibrio sp. 1195163 1212198 289208308/ 289208309/ TK90_1129/ 289208312/ 289208315/ 289208316/ K90mix 6225 6227 6229 6231 6233 6235 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 11 BREX type 5 systems Genomic Genomic Start End Organism Point Point brxA* brxB* BrxC/pglY* pglX* pglZ* brxHII* Halopiger xanaduensis SH6 275443 305942 336252375/ 238756192/ 336252376/ 336252387/ 336252390/ 336252392/ uid68105 5065 3007 736 2917 1835 3498 336252378/ 737 Halorhabdus utahensis DSM 1919731 1943689 missing 238756192/ 257052978/ 257052985/ 257052987/ 257052989/ 12940 uid59189 3008 738 2918 1836 3499 257052980/ 739 Halobacterium salinarum R1 217533 239329 169237558/ 238756192/ 169237555/ 169237551/ 169237549/ missing uid61571 5066 3009 740 2919 1837 169237557/ 741 Halorubrum lacusprofundi 421881 464896 222476096/ 238756192/ 222476093/ 222476090/ 222476089/ 222476088/ ATCC 49239 uid58807 5067 3010 742 2920 1838 3500 222476095/ 222476117/ 743 2921 Haloarcula hispanica ATCC 401471 426563 344209887/ 238756192/ 344209884/ 344209878/ 344209879/ 344209877/ 33960 uid72475 5068 3011 744 2922 1839 3501 344209886/ 344209881/ 745 2923 halophilic archaeon DL31 245839 265734 345007035/ 238756192/ 345007036/ 345007041/ 345007043/ 345007044/ uid72619 5069 3012 746 2924 1840 3502 345007038/ 747 Natrinema pellirubrum DSM 247457 267369 433593286/ 238756192/ 433593287/ 433593291/ 433593294/ 433593295/ 15624 uid74437 5070 3013 748 2925 1841 3503 433593289/ 433593292/ 749 2926 Natronorubrum tibetense 16416 35521 448302571/ 238756192/ 448302568/ missing 448302567/ 448302575/ GA33 5368 3014 1111 2193 3527 448302570/ 1112 Haloarcula argentinensis 133870 160866 448682081/ 238756192/ 448682078/ 448682072/ 448682071/ 448682070/ DSM 12282 5369 3015 1113 3252 2194 3528 448682080/ 1114 Halosimplex carlsbadense 2- 45427 67411 445667054/ 238756192/ 448413236/ missing 448413226/ 448413225/ 9-1 6167 3016 1115 2195 3529 448413238/ 1116 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 12 BREX type 6 systems Genomic Genomic Start End Organism Point Point brxE* brxA* brxB* Planctomyces limnophilus 3979304 3995634 296123323/ 296123322/ 296123321/ DSM 3776 uid48643 6037 5071 5694 Anaeromyxobacter 1284321 1301040 220916259/ 220916260/ 220916261/ dehalogenans 2CP 1 6038 5072 5695 uid58989 Haliangium ochraceum 798493 815906 262193918/ 262193919/ 262193920/ DSM 14365 uid41425 6039 5073 5696 Haliangium ochraceum 1611313 1628687 262194477/ 262194478/ 262194479/ DSM 14365 uid41425 6040 5074 5697 Rhodopirellula 94858 111243 470888969/ 470888968/ 470888967/ sp. SWK7 6035 5370 5702 Pseudanabaena biceps 10824 29281 443475075/ 443475074/ 443475073/ PCC 7429 6036 5371 5943 Organism BrxC/pglY* pglX* pglZ* brxD* brxHI* Planctomyces limnophilus 296123320/ 296123319/ 296123318/ 296123317/ 296123316/ DSM 3776 uid48643 750 2927 1842 4441 3622 Anaeromyxobacter 220916262/ 220916263/ 220916264/ 220916266/ 220916267/ dehalogenans 2CP 1 751 2928 1843 4442 3623 uid58989 Haliangium ochraceum 262193921/ 262193922/ 262193923/ 262193924/ 262193925/ DSM 14365 uid41425 752 2929 1844 4443 3624 Haliangium ochraceum 262194480/ 262194481/ 262194482/ 262194483/ 262194484/ DSM 14365 uid41425 753 2930 1845 4444 3625 Rhodopirellula 470888966/ 470888965/ 470888964/ 470888963/ 470888962/ sp. SWK7 1117 6186 2196 4464 3626 Pseudanabaena biceps 443475072/ 443475069/ 443475065/ 443475063/ 443475062/ PCC 7429 1118 6188 2197 4465 3627 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 13 BREX type 3 systems Genomic Genomic Start End Organism Point Point brxF* brxC/pglY* pglXI* brxHII* pglZ* brxA* Acetohalobium arabaticum 1471809 1481230 302392145/ 302392144/ 302392143/ missing 302392142/ 302392141/ DSM 5501 uid51423 5919 754 3343 1846 5075 Acidothermus 895934 911157 117928019/ 117928020/ 117928022/ 117928024/ 117928025/ 117928026/ cellulolyticus 11B 5980 755 3344 3504 1847 5076 uid58501 Anaerobaculum mobile 1894751 1909131 392408180/ 392408179/ 392408178/ 392408176/ 392408175/ 392408174/ DSM 13181 uid168323 5981 756 3345 3505 1848 5077 Bacteroides vulgatus 4615002 4627784 missing 150006230/ 150006231/ 150006233/ 150006234/ 150006235/ ATCC 8482 uid58253 757 3346 3506 1849 5078 Caldicellulosiruptor 671655 683698 312792856/ 312792857/ 312792858/ Calkr_0625/ 312792859/ 312792860/ kristjanssonii 177R1B 5982 758 3347 6173 1850 5079 uid60393 Chloroflexus aggregans 1376256 1391910 219848031/ 219848033/ 219848032/ 219848036/ 219848037/ 219848038/ DSM 9485 uid58621 5983 759 3348 3507 1853 5080 219848035/ 3349 Desulfovibrio aespoeensis 1822100 1838149 317153363/ 317153364/ 317353366/ 317153368/ 317153369/ 317153370/ Aspo 2 uid42613 5984 760 3350 3508 1852 5081 Dichelobacter nodosus 185801 200163 146328698/ 146329396/ 146329329/ 146329165/ 146329856/ 146328877/ VCS1703A uid57643 5985 761 3351 3509 1853 5082 Methanocaldococcus 892177 917259 289192374/ 289192375/ 289192376/ missing 289192377/ 289192378/ FS406 22 uid42499 5986 762 3352 1854 5083 Methanosalsum zhilinae 1398867 1423843 336477236/ 336477235/ 336477234/ 336477230/ 336477229/ 336477228/ DSM 4017 uid68249 5987 764 3353 3510 1855 5084 336477237/ 3354 Methylacidiphilum 315550 333912 189218342/ 189218341/ 189218335/ 189218338/ 189218337/ 189218336/ infernorum V4 uid59161 5988 765 3355 3511 1856 5085 189218340/ 3356 Nitrosococcus oceani 53559 70307 77163602/ 77163603/ 77163606/ 77163610/ 77163611/ 77163612/ ATCC 19707 uid58403 5990 767 3358 3513 1858 5087 Nitrosococcus watsonii C 43703 58760 300112781/ 300112782/ 300112784/ 300112788/ 300112789/ 300112790/ 113 uid50331 5991 768 3359 3514 1859 5088 Parvibaculum 1304571 1320948 154251635/ 154251634/ 154251631/ 154251630/ 154251629/ 154251628/ lavamentivorans DS 1 5992 769 3360 3515 1860 5089 uid58739 Parvibaculum 3796620 3812997 154253985/ 154253986/ 154253989/ 154253990/ 154253991/ 154253992/ lavamentivorans DS 1 5993 770 3361 3516 1861 5090 uid58739 Planctomyces brasiliensis 1319340 1355964 325107713/ 325107712/ 325107710/ 325107709/ 325107708/ 325107707/ DSM 5305 uid60583 5994 771 3362 3517 1862 5091 Syntrophothermus 1307414 1328282 297617453/ 297617452/ 297617451/ missing 297617450/ 297617449/ lipocalidus DSM 12680 5995 772 3363 1863 5092 uid49527 Tepidanaerobacter 1934820 1944222 438003074/ 438003073/ 438003072/ missing 438003071/ 438003070/ acetatoxydans Re1 5996 774 3364 1864 5093 uid184827 Tepidanaerobacter Re1 1933717 1943118 332799810/ 332799809/ 332799808/ missing 332799807/ 332799806/ uid66873 5997 775 3365 1865 5094 Thermanaerovibrio 1551823 1568220 269793100/ 269793099/ 269793097/ 269793095/ 269793094/ 269793093/ acidaminovorans DSM 5998 776 3366 3518 1866 5095 6589 uid41925 Thermoanaerobacter 974096 986151 320115754/ 320115755/ 320115756/ 320115757/ 320115758/ 320115759/ brockii finnii Ako 1 5999 777 3367 3519 1867 5096 uid55639 Thermoanaerobacter 1369765 1379419 289578546/ 289578545/ 289578544/ missing 289578542/ 289578541/ italicus Ab9 uid46241 6000 778 3368 1868 5097 Thermoanaerobacter 981903 993958 167037338/ 167037339/ 167037340/ 167037341/ 167037342/ 167037343/ pseudethanolicus ATCC 6001 779 3369 3520 1869 5098 33223 uid58339 Thermoanaerobacterium 2325486 2337569 390935378/ 390935377/ 390935376/ 390935375/ 390935374/ 390935373/ saccharolyticum JW SL 6002 780 3370 3521 1870 5099 YS485 uid167781 Thermoanaerobacterium 1846040 1858147 433655487/ 433655486/ 433655485/ 433655484/ 433655483/ 433655482/ thermosaccharolyticum 6003 781 3371 3522 1871 5100 M0795 uid184821 Thermoanaerobacterium 1036830 1048931 333896805/ 333896806/ 333896807/ 333896808/ 333896809/ 333896810/ xylanolyticum LX 11 6004 782 3372 3523 1872 5101 uid63163 Pelotomaculum 698030 719373 147677041/ 147677050/ #N/A 147677052/ thermopropionicum SI 616 3403 1177 uid58877 planctomycete KSU-1 302255 318802 386811047/ 386811046/ 386811042/ 386811038/ 386811037/ 386811036/ 6005 1119 3373 3530 2198 5372 Bacillus cereus W 64027 76920 196035241/ 196035162/ 196035235/ 196035256/ 196035132/ 196035248/ 6006 1120 3374 3531 2199 5373 Clostridium thermocellum 67367 77609 missing 419725844/ 419725843/ 419725842/ 419725841/ 419725839/ YS 1121 3375 3532 2200 5374 419725840/ 5375 Lachnospiraceae bacteriu 835791 847349 336426893/ 336426892/ 336426890/ 336426889/ 336426888/ 336426887/ 6007 1122 3376 3533 2201 5376 336426891/ 3377 Enterococcus faecium 115722 257885933/ 257885934/ 257885935/ missing 257885936/ 257885937/ 1,231,501 6008 1123 3378 2202 6170 Caloramator australicus 17570 34190 397905665/ 397905666/ 397905667/ 397905670/ 397905671/ 397905672/ RC3]Length = 15 6009 1124 3379 3534 2203 5377 397905668/ 3380 397905669/ 3381 397905678/ 3382 Pseudoalteromonas marina 51753 65055 392538886/ 392538885/ 392538884/ 392538883/ 392538882/ 392538880/ mano4 6010 1125 3383 3535 2204 5378 392538881/ 5379 Desulfotomaculum 104421 114874 323701233/ 323701234/ 323701235/ missing 323701238/ 323701239/ nigrificans DSM 574 6011 1126 3384 2205 5380 Dethiosulfovibrio 2471763 2489488 288575103/ 288575102/ 288575100/ DpepDRAFT_(—) 288575095/ 288575094/ peptidovorans DSM 11002 6012 1127 3385 2372/6176 2206 5381 Kingella denitrificans 998416 1009424 325266722/ 325266723/ 325266724/ 325266725/ 325266726/ 325266727/ ATCC 33394 6013 1128 3386 6177 2207 5382 Alcanivorax 66342 85539 408373946/ 408373945/ 408373941/ 408373939/ 408373938/ 408373937/ hongdengensis A-11-3 6014 1129 3387 3536 2208 5383 Bacillus cereus BAG2X1- 4134446 4144054 423398560/ 423398559/ 423398558/ 401647033/ 423398557/ 423398556/ 1 6015 1130 3388 6178 2209 5384 Nitrosococcus oceani 570876 587579 254435536/ 254435628/ 254435024/ missing 254436183/ 254435793/ AFC27 6016 1131 3389 2210 5385 Methyloversatilis 13404 25532 missing 334130736/ 334130735/ 334130734/ 334130733/ 334130732/ universalis FAM5 1132 3390 3537 2211 5386 Thermoplasmatales 1730 14243 472439489/ 472439490/ 472439492/ missing 472439494/ 472439495/ archaeon SCGC AB-539- 6017 1133 3391 2212 5387 N05 Pseudomonas sp. GM55 71736 85194 398889060/ 398889059/ 398889058/ PMI31_00817 398889056/ 398889055/ 6018 1134 3392 2213 5388 Bacillus cereus 03BB108 37106 46755 196044400/ 196044278/ 196044173/ 168166142 196044220/ 196044133/ 6019 1135 3393 2214 5389 Treponema primitia ZAS- 9000 34287 374812240/ 374812241/ 374812242/ 374812236/ 374812248/ 374812249/ 1 6020 1136 3394 3538 2215 5415 Bacillus methanolicus 39939 49733 415887046/ 415887047/ 415887048/ MGA3_16723/ 415887049/ 415887050/ MGA3 6021 1137 3395 6180 2216 5390 Vibrio scophthalmi LMG 609 13898 343509541/ 343509540/ 343509539/ 343509538/ 343509537/ 343509534/ 19158 6022 1138 3396 3539 2217 5391 343509535/ 5392 343509536/ 5393 Thiorhodovibrio sp. 970 32332 48989 381156867/ 381156866/ 381156864/ Thi970DRAFT_(—) 381156859/ 381156858/ 6023 1139 3397 0448/6183 2218 5394 Ectothiorhodospir 1 13805 374623711/ 374623710/ 374623709/ 374623707/ 374623706/ missing 6024 1140 3398 3540 2219 Clostridium papyrosolvens 27432 39463 326204497/ 326204498/ 326204499/ 326204500/ 326204501/ 326204502/ DSM 2782 6025 1141 3399 3541 2220 5395 Thermoanaerobacter 35382 49116 326389944/ 326389943/ 326389941/ 326389940/ 326389939/ 326389938/ ethanolicus JW 200 6026 1142 3400 3542 2221 5396 Thermoanaerobacter 8992 18633 256752453/ 256752454/ 256752456/ missing 256752458/ 256752459/ ethanolicus CCSD1 6027 1143 3401 2222 5397 256752455/ 1144 Nitrococcus mobilis Nb- 2181486 2199782 88811251/ 88811250/ 88811247/ NB231_10583/ 88811242/ 88811241/ 231 6028 1145 3402 6184 2223 5398 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 14 BREX type 2 systems Genomic Genomic Start End Organism Point Point pglW* pglX* pglY* pglZ* brxD* brxHI* Burkholderia thailandensis 131918 145741 83720701/ 83719870/ 83719623/ 83719262/ missing missing E264 uid58081 6091 2931 783 1873 Candidatus Accumulibacter 3869550 3890263 257094932/ 257094931/ 257094926/ 257094925/ 257094924/ 257094923/ phosphalis clade IIA UW 1 6092 2932 784 1874 4445 3596 uid59207 Corallococcus coralloides 6591319 6611764 383457207/ 383457206/ 383457202/ 383457203/ missing 383457201/ DSM 2259 uid157997 6093 2933 785 1875 3597 383457204/ 786 Corynebacterium variabile 1913373 1933608 340794635/ 340794636/ 340794637/ 340794638/ 340794639/ 340794640/ DSM 44702 uid62003 6094 2934 787 1876 4446 3598 Frankia CcI3 uid58397 3489708 3507585 86741639/ 86741640/ 86741641/ 86741642/ 86741643/ 86741644/ 6095 2935 788 1877 4447 3599 Frankia EuI1c uid42615 6951904 6971263 312199458/ 312199459/ 312199460/ 312199461/ 312199462/ 312199463/ 6096 2936 789 1878 4448 3600 Haliangium ochraceum DSM 1565170 1582937 262194452/ 262194453/ 262194454/ 262194455/ 262194456/ 262194457/ 14365 uid41425 6097 2937 790 1879 791 3601 Microlunatus phosphovorus 3075705 3093432 336118511/ 336118510/ 336118509/ 336118508/ 336118507/ 336118506/ NM 1 uid68055 6098 2938 792 1880 4449 3602 Micromonospora aurantiaca 1329830 1350410 302865792/ 302865793/ 302865796/ 302865797/ 302865798/ 302865799/ ATCC 27029 uid42501 6099 2939 793 1881 4450 3603 Mycobacterium gilvum PYR 3386977 3404461 145223791/ 145223790/ 145223789/ 145223788/ 145223787/ 145223786/ GCK uid59421 6100 2940 794 1882 4451 3604 Nocardia cyriacigeorgica 1974963 1993641 379708002/ 379708001/ 379708000/ 379707999/ 379707998/ 379707997/ GUH 2 uid89395 6101 2941 795 1883 4452 3605 Polaromonas 170793 210985 121582867/ 121582865/ 121582862/ 121582861/ 121582860/ 121582859/ naphthalenivorans CJ2 6102 2942 796 1884 4453 3606 uid58273 121582883/ 2943 Saccharomonospora viridis 508003 530821 257054581/ 257054590/ 257054591/ 257054592/ 257054593/ 257054594/ DSM 43017 uid59055 6103 2944 797 1885 4454 3607 Saccharopolyspora erythraea 5714083 5731753 134101641/ 134101640/ 134101639/ 134101638/ 134101637/ 134101636/ NRRL 2338 uid62947 6104 2945 798 1886 4455 3608 Saccharothrix espanaensis 1882970 1907934 433603667/ 433603658/ 433603653/ 433603652/ 433603651/ 433603650/ DSM 44229 uid184826 6105 2946 799 1887 4456 3609 Singulisphaera acidiphila 2421532 2440518 430742885/ 430742884/ 430742881/ 430742880/ 430742879/ 430742878/ DSM 18658 uid81777 6106 2947 800 1888 4457 3610 Sorangium cellulosum So ce 10676864 10731426 162455957/ 162455958/ 162455964/ 162455967/ 162455968/ 162455969/ 56 uid61629 6107 2948 801 1889 4458 3611 162455970/ 6108 162455983/ 6109 Streptomyces coelicolor A3 2 7348537 7376403 21224924/ 32141309/ 21224932/ 21224933/ 21224936/ 21224937/ uid57801 6110 2949 802 1890 4459 3612 Streptomyces griseus NBRC 1877109 1900853 182435393/ 182435394/ 182435399/ 182435400/ 182435403/ 182435404/ 13350 uid58983 6111 2950 803 1891 4460 3613 Thermobifida fusca YX 810381 853382 72161105/ 72161128/ 72161113/ 72161114/ 72161115/ 72161116/ uid57703 6112 2951 804 1892 4461 3614 Thermobispora bispora DSM 1764882 1779583 296269520/ 296269521/ 296269522/ 296269523/ missing missing 43833 uid48999 6113 2952 805 1893 Hahella chejuensis KCTC 3587257 3606877 83646207/ 83646204/ 83646203/ 83646202/ 83646201/ 83646200/ 2396 uid58483 6114 2953 806 1894 4462 3615 Saccharomonospora glauca 539845 562776 384564483/ 384564490/ 384564491/ 384564492/ 384564493/ 384564494/ K62 6115 3253 1146 2224 4466 3616 Rhodococcus triatomae BKS 58688 79518 453074717/ 453074718/ 453074722/ 453074723/ 453074724/ 453074725/ 15-14 6116 3254 1147 2225 4467 3617 Saccharomonospora cyanea 604367 625835 375098936/ 375098940/ 375098941/ 375098942/ 375098943/ 375098944/ NA-134 6117 3255 1148 2226 4468 3618 Gordonia polyisoprenivorans 51878 69760 359765504/ 359765505/ 359765506/ 359765507/ 359765508/ 359765509/ NBRC 16320 6118 3256 1149 2227 4469 3619 Amycolatopsis azurea DSM 30190 53418 451335443/ 451335436/ 451335435/ 451335434/ 451335432/ 451335431/ 43854 6119 3257 1150 2228 4470 3620 Marinobacter sp. ELB17 49994 72968 126666550/ 126666547/ 126666537/ 126666536/ 126666535/ 126666534/ 6120 3258 1151 2229 4471 3621 Saccharopolyspora erythraea 144893 160200 291003191/ 291003192/ 291003193/ 291003194/ 291003195/ missing NRRL 2338 6121 3259 1152 2230 4472 Streptomyces turgidiscabies 9173 41654 440700787/ 440700766/ 440700790/ 440700796/ 440700764/ 440700770/ Car8 6122 3260 1153 2231 4473 3628 Gemma taobscuriglobus 27609 48046 168697904/ 168697902/ 168697899/ 168697898/ 168697897/ 168697896/ UQM 2246 6123 3261 1154 2232 4474 3629 Rhodococcus ruber BKS 20- 6599 29593 458780602/ 458780601/ 458780598/ 458780597/ 458780596/ 458780593/ 38 6124 3262 1155 2233 4475 3630 Mycobacterium 216677 234966 383824736/ 383824739/ 383824740/ 383824741/ 383824742/ 383824743/ xenopi RIVM700367 6125 3263 1156 2234 4476 3631 Micromonospora sp. ATCC 4955630 4982819 238063210/ 238063211/ 238063221/ 238063222/ 238063223/ 238063224/ 39149 6126 3264 1157 2235 4477 3632 Saccharomonospora 1696320 1718030 383828993/ 383828987/ 383828986/ 383828985/ 383828984/ 383828983/ xinjiangensis XJ-54 6127 3265 1158 2236 4478 3633 Bradyrhizobium sp. ORS 375 19316 37325 365878962/ 365878961/ 365878960/ 365878959/ 365878958/ 365878957/ 6128 3266 1159 2237 4479 3634 Burkholderia thailandensis 3406043 3419866 257140682/ 257140680/ 257140679/ 257140678/ missing missing E264 6129 3267 1160 2238 Planctomyces maris DSM 100676 119360 149176313/ 149176311/ 149176310/ 149176309/ 149176308/ 149176307/ 8797 6130 3268 1161 2239 4480 3635 Streptomyces gancidicus BKS 67474 97086 458859650/ 458859651/ 458859660/ 458859661/ 458859664/ 458859665/ 13-15 6131 3269 1162 2240 4481 3636 Gordonia amicalis NBRC 3958 21621 441515888/ 441515889/ 441515890/ 441515891/ 441515892/ 441515893/ 100051 = JCM 11271 6132 3270 1163 2241 4482 3637 Mycobacterium intracellulare 14963 30468 254821509/ 254821510/ 254821511/ 254821513/ 254821514/ missing ATCC 13950 6133 3271 1164 2242 4483 Phaeospirillum molischianum 9727 29750 381168760/ 381168763/ 381168766/ 381168767/ 381168768/ 381168769/ DSM 120 6134 3272 1165 2243 4484 3638 Nitrococcus mobilis Nb-231 3284658 3304962 88810583/ 88810586/ 88810589/ 88810590/ 88810591/ 88810592/ 6135 3273 1166 2244 4485 3639 88810585/ 6136 Frankia sp. EUN1f 4751 24481 288920048/ 288920049/ 288920050/ 288920051/ 288920052/ 288920053/ 6137 3274 1167 2245 4486 3640 Pseudomonas stutzeri NF13 77221 95110 452746641/ missing 452746643/ 452746644/ 452746645/ 452746646/ 6138 1168 2246 4487 3641 Dietzia cinnamea P4 1 10875 missing 319947886/ 319947887/ 319947888/ 319947889/ 319947890/ 3275 1169 2247 4488 3642 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 15 BREX type 4 systems Genomic Genomic Start End Organism Point Point brxP* brxC/pglY* pglZ* brxL* Aciduliprofundum MAR08 1211535 1229434 432329235/ 432329234/ 432329233/ 432329232/ 339 uid184407 3428 807 1895 4403 Anaerobaculum mobile DSM 1574689 1585597 392407896/ 392407895/ 392407894/ 392407893/ 13181 uid168323 3429 808 1896 4404 392407897/ 3430 Candidatus Desulforudis 837185 861804 169830967/ 169830968/ 169830970/ 169830971/ audaxviator MP104C 3431 809 1897 4405 uid59067 Coprothermobacter 1373786 1384972 206895424/ 206896068/ 206895399/ 206895834/ proteolyticus DSM 5265 3432 810 1898 4406 uid59253 206895920/ 3433 Cyanobacterium stanieri PCC 2847921 2863377 428774403/ 428774405/ 428774399/ 428774398/ 7202 uid183337 3434 812 1899 4407 Denitrovibrio acetiphilus 583467 593592 291286509/ 291286510/ 291286511/ 291286512/ DSM 12809 uid46657 3435 813 1900 4408 Desulfitobacterium 793871 804051 431792781/ 431792782/ 431792783/ 431792784/ dichloroeliminans LMG P 3436 814 1901 4409 21439 uid82555 Geobacter M21 uid59037 937933 948073 253699433/ 253699434/ 253699435/ 253699436/ 3437 815 1902 4410 Prevotella denticola F0289 414128 424374 327312660/ 327312662/ 327312663/ 327312664/ uid65091 3438 816 1903 4411 Thermotoga petrophila RKU 1747153 1782820 148270868/ 148270869/ 148270870/ 148270871/ 1 uid58655 3439 817 1904 4412 Thermomicrobium roseum 91162 104624 A truncated 221635509/ 221635513/ 221635514/ DSM 5159 uid59341 brxP was 1714 1714 4299 missed by ORF finder tool. *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 16 Summary of distribution of BREX types across genomes BREX BREX BREX BREX BREX BREX Organism #1 #5 #6 #3 #2 #4 Comments 1 Pseudanabaena biceps PCC 7429 x x Genome contains two BREX systems: #1 and #6 2 Moorella thermoacetica ATCC 39073 x uid58051 3 Tepidanaerobacter acetatoxydans Re1 x x Genome contains two BREX uid184827 systems: #1 and #3 4 Tepidanaerobacter Re1 uid66873 x x Genome contains two BREX systems: #1 and #3 5 Thiorhodovibrio sp. 970 x x Genome contains two BREX systems: #1 and #3 6 Marinobacter sp. ELB17 x x Genome contains two BREX systems: #1 and #2 7 Microlunatus phosphovorus NM 1 uid68055 x x Genome contains two BREX systems: #1 and #2 8 Acetivibrio cellulolyticus CD2 x 9 Acidiphilium multivorum AIU301 uid63345 x 10 Acidithiobacillus ferrivorans SS3 uid67387 x 11 Acidovorax sp. NO-1 x 12 Acinetobacter baumannii AB5256 x 13 Acinetobacter baumannii AYE uid61637 x 14 Acinetobacter baumannii Naval-18 x 15 Acinetobacter baumannii OIFC098 x 16 Acinetobacter baumannii WC-136 x 17 Acinetobacter johnsonii SH046 x 18 Acinetobacter junii SH205 x 19 Acinetobacter lwoffii SH145 x 20 Acinetobacter radioresistens DSM 6976 = x NBRC 102413 21 Acinetobacter sp. P8-3-8 x 22 Acinetobacter sp. SH024 x 23 Actinomyces neuii BVS029A5 x 24 Actinomyces sp. ICM47 x 25 Actinomyces sp. oral taxon 178 str x 26 Alcanivorax pacificus W11-5 x 27 Alcanivorax sp. DG881 x 28 Alteromonas macleodii Black Sea 11 x uid176365 29 Anaeromyxobacter dehalogenans 2CP C x uid58135 30 Aromatoleum aromaticum EbN1 uid58231 x 31 Arthrobacter nitroguajacolicus Rue61a x uid174511 32 Aurantimonas manganoxydans SI85-9A1 x 33 Azospirillum lipoferum 4B uid82343 x 34 Bacillus cereus BAG6X1-2 x 35 Bacillus cereus H3081.97 x 36 Bacillus cereus HuB2-9 x 37 Bacteroides coprophilus DSM 18228 x 38 Bacteroides ovatus CL02T12C04 x 39 Bacteroides ovatus SD CC 2a x 40 Bacteroides ovatus SD CMC 3f x 41 Bacteroides sp. 1_1_14 x 42 Bacteroides sp. 2_1_7 x 43 Bacteroides sp. 3_1_33FAA x 44 Bacteroides sp. D1 x 45 Bacteroides sp. D2 x 46 Bacteroides sp. 2_1_22 x 47 Bacteroides xylanisolvens SD CC 1b x 48 Bifidobacterium angulatum DSM 20098 x 49 Bifidobacterium animalis ATCC 25527 x uid162513 50 Bifidobacterium bifidum IPL x 51 Bifidobacterium bifidum NCIMB 41171 x 52 Bordetella parapertussis Bpp5 uid177516 x 53 Brachybacterium paraconglomeratum LC44 x 54 Brevibacterium mcbrellneri ATCC 49030 x 55 Burkholderia CCGE1001 uid42975 x 56 Burkholderia gladioli BSR3 uid66301 x 57 Burkholderia vietnamiensis G4 uid58075 x 58 Calditerrivibrio nitroreducens DSM 19672 x uid60821 59 Carboxydothermus hydrogenoformans Z x 2901 uid57821 60 Cellvibrio sp. BR x 61 Chlorobium phaeobacteroides BS1 x uid58131 62 Clostridium butyricum E4 str. BoNT E x BL5262 63 Clostridium ljungdahlii DSM 13528 x uid50583 64 Clostridium perfringens C str. JGS1495 x 65 Clostridium perfringens D str. JGS1721 x 66 Clostridium sticklandii DSM 519 uid59585 x 67 Clostridium SY8519 uid68705 x 68 Collinsella aerofaciens ATCC 25986 x 69 Collinsella intestinalis DSM 13280 x 70 Collinsella stercoris DSM 13279 x 71 Coprobacillus sp. 3_3_56FAA x 72 Coprobacillus sp. 8_2_54BFAA x 73 Cupriavidus necator N 1 uid68689 x 74 Cyanothece PCC 8802 uid59143 x 75 Cylindrospermopsis raciborskii CS-505 x 76 Dehalococcoides VS uid42393 x 77 Dehalogenimonas lykanthroporepellens BL x DC 9 uid48131 78 delta proteobacterium NaphS2 x 79 Desulfitobacterium hafniense Y51 uid58605 x 80 Desulfobacula toluolica Tol2 uid175777 x 81 Desulfomicrobium baculatum DSM 4028 x uid59217 82 Desulfonatronospira thiodismutans ASO3-1 x 83 Desulfosporosinus meridiei DSM 13257 x uid75097 84 Desulfotomaculum gibsoniae DS x 85 Desulfovibrio magneticus RS 1 uid59309 x 86 Desulfovibrio vulgaris Hildenborough x uid57645 87 Desulfovibrio vulgaris RCH1 uid161961 x 88 Desulfurivibrio alkaliphilus AHT2 uid49487 x 89 Enterobacter cloacae ENHKU01 uid172463 x 90 Enterobacter hormaechei ATCC 49162 x 91 Enterobacter sp. SST3 x 92 Enterococcus faecalis TX0109 x 93 Enterococcus faecalis TX1302 x 94 Enterococcus faecium 509 x 95 Enterococcus faecium 511 x 96 Enterococcus faecium 514 x 97 Enterococcus faecium C1904 x 98 Enterococcus faecium C497 x 99 Enterococcus faecium E0679 x 100 Enterococcus faecium E1731 x 101 Enterococcus faecium E1904 x 102 Enterococcus faecium E2883 x 103 Enterococcus faecium ERV99 x 104 Enterococcus faecium P1123 x 105 Enterococcus faecium P1137 x 106 Enterococcus faecium P1139 x 107 Enterococcus faecium TX0133A x 108 Enterococcus faecium TX0133a01 x 109 Enterococcus faecium TX0133a04 x 110 Enterococcus faecium TX0133B x 111 Enterococcus faecium TX0133C x 112 Erwinia Ejp617 uid159955 x 113 Erwinia pyrifoliae DSM 12163 uid159693 x 114 Erwinia pyrifoliae Ep1 96 uid40659 x 115 Erythrobacter litoralis HTCC2594 uid58299 x 116 Escherichia coli clone D i14 uid162049 x 117 Escherichia coli clone D i2 uid162047 x 118 Escherichia coli 2534-86 x 119 Escherichia coli 3.3884 x 120 Escherichia coli 4.0522 x 121 Escherichia coli 96.154 x 122 Escherichia coli B41 x 123 Escherichia coli B799 x 124 Escherichia coli DEC10E x 125 Escherichia coli DEC10F x 126 Escherichia coli DEC13A x 127 Escherichia coli DEC13B x 128 Escherichia coli DEC13C x 129 Escherichia coli DEC13D x 130 Escherichia coli DEC13E x 131 Escherichia coli DEC14B x 132 Escherichia coli DEC14C x 133 Escherichia coli DEC14D x 134 Escherichia coli DEC7B x 135 Escherichia coli DEC8A x 136 Escherichia coli DEC8B x 137 Escherichia coli DEC9A x 138 Escherichia coli DEC9B x 139 Escherichia coli DEC9C x 140 Escherichia coli DEC9D x 141 Escherichia coli DEC9E x 142 Escherichia coli HS uid58393 x 143 Escherichia coli JB1-95 x 144 Escherichia coli KD2 x 145 Escherichia coli KTE12 x 146 Escherichia coli KTE139 x 147 Escherichia coli KTE153 x 148 Escherichia coli KTE211 x 149 Escherichia coli KTE218 x 150 Escherichia coli KTE234 x 151 Escherichia coli KTE47 x 152 Escherichia coli KTE53 x 153 Escherichia coli KTE6 x 154 Escherichia coli MS 69-1 x 155 Escherichia coli O10:K5(L):H4 str. ATCC x 23506 156 Escherichia coli O111 H 11128 uid41023 x 157 Escherichia coli O111:H8 str. CVM9570 x 158 Escherichia coli O111:H8 str. CVM9574 x 159 Escherichia coli O111:H8 str. CVM9602 x 160 Escherichia coli O111:H8 str. CVM9634 x 161 Escherichia coli O113:H21 str. CL-3 x 162 Escherichia coli O25b:ST131 str. JIE186 x 163 Escherichia coli OK1180 x 164 Escherichia coli S17 x 165 Escherichia coli STEC_94C x 166 Escherichia coli TW10828 x 167 Escherichia fergusonii ATCC 35469 x uid59375 168 Eubacterium cellulosolvens 6 x 169 Exiguobacterium sibiricum 255 15 uid58053 x 170 Faecalibacterium prausnitzii A2-165 x 171 Faecalibacterium prausnitzii M21/2 x 172 Flavobacterium branchiophilum FL 15 x uid73421 173 Fulvimarina pelagi HTCC2506 x 174 Fusobacterium nucleatum subsp. nucleatum x ATCC 23726 175 Fusobacterium sp. 11_3_2 x 176 Fusobacterium sp. 2_1_31 x 177 Fusobacterium sp. 3_1_27 x 178 Fusobacterium sp. 3_1_5R x 179 Fusobacterium sp. 7_1 x 180 Fusobacterium ulcerans ATCC 49185 x 181 gamma proteobacterium BDW918 x 182 gamma proteobacterium IMCC3088 x 183 Geobacillus sp. G11MC16 x 184 Geobacillus thermoglucosidan x 185 Geobacillus WCH70 uid59045 x 186 Geobacter sulfurreducens PCA uid57743 x 187 Glaciecola lipolytica E3 x 188 Glaciecola polaris LMG 21857 x 189 Glaciecola punicea DSM 14233 = ACAM x 611 190 Haliscomenobacter hydrossis DSM 1100 x uid66777 191 Haloarcula japonica DSM 6131 x 192 Halobacillus halophilus DSM 2266 x uid162033 193 Halobacteroides halobius DSM 5150 x uid184862 194 Halomonas sp. HAL1 x 195 Holdemania filiformis DSM 12042 x 196 Holophaga foetida DSM 6591 x 197 Johnsonella ignava ATCC 51276 x 198 Klebsiella oxytoca E718 uid170256 x 199 Lachnoanaerobaculum (Eubacterium) x saburreum DSM 3986 200 Lachnoanaerobaculum (Eubacterium) x saburreum F0468 201 Lachnospiraceae bacterium 1_1_57FAA x 202 Lachnospiraceae bacterium 8_1_57FAA x 203 Lachnospiraceae bacterium oral taxon 082 x str 204 Lachnospiraceae oral taxon 107 str x 205 Lactobacillus amylovorus GRL1118 x uid160233 206 Lactobacillus casei Lpc-37 x 207 Lactobacillus casei UW4 x 208 Lactobacillus casei Zhang uid50673 x 209 Lactobacillus helveticus DSM 20075 x 210 Lactobacillus helveticus H10 uid162017 x 211 Lactobacillus helveticus R0052 uid174439 x 212 Lactobacillus johnsonii ATCC 33200 x 213 Lactobacillus johnsonii FI9785 uid41735 x 214 Lactobacillus reuteri CF48-3A x 215 Lactobacillus reuteri SD2112 uid55357 x 216 Lactobacillus rhamnosus GG uid161983 x 217 Lactobacillus rhamnosus GG uid59313 x 218 Lactobacillus rhamnosus LRHMDP2 x 219 Lactobacillus rhamnosus LRHMDP3 x 220 Lactobacillus zeae KCTC 3804 x 221 Leuconostoc kimchii IMSNU 11154 x uid48589 222 Magnetospirillum magneticum AMB 1 x uid58527 223 Marinobacter aquaeolei VT8 uid59419 x 224 Methanobrevibacter smithii ATCC 35061 x uid58827 225 Methanoculleus bourgensis MS2 uid171377 x 226 Methanolobus psychrophilus R15 x uid177925 227 Methanomethylovorans hollandica DSM x 15978 uid184864 228 Methanosarcina acetivorans C2A uid57879 x 229 Methanosarcina mazei Go1 uid57893 x 230 Methylophaga aminisulfidivorans MP x 231 Microcystis aeruginosa PCC 9443 x 232 Nitratireductor indicus C115 x 233 Nostoc punctiforme PCC 73102 uid57767 x 234 Opitutacea x 235 Oribacterium sp. ACB1 x 236 Oribacterium sp. ACB7 x 237 Oribacterium sp. ACB8 x 238 Paenibacillus elgii B69 x 239 Pantoea sp. GM01 x 240 Parabacteroides sp. D25 x 241 Parvularcula bermudensis HTCC2503 x uid51641 242 Pectobacterium carotovorum PCC21 x uid174335 243 Pectobacterium wasabiae CFBP 3304 x 244 Pelobacter propionicus DSM 2379 uid58255 x 245 Pelodictyon phaeoclathratiforme BU 1 x uid58173 246 Photobacterium sp. SKA34 x 247 Photorhabdus asymbiotica uid59243 x 248 Polaromonas JS666 uid58207 x 249 Proteus mirabilis WGLW6 x 250 Pseudoalteromona x 251 Pseudoalteromonas luteoviolacea B = x ATCC 29581 252 Pseudomonas brassicacearum NFM421 x uid66303 253 Pseudomonas fragi A22 x 254 Pseudomonas fuscovaginae UPB0736 x 255 Pseudomonas mandelii JR-1 x 256 Pseudomonas psychrotolerans L19 x 257 Pseudomonas stutzeri CCUG 29243 x uid168379 258 Pseudomonas syringae Lz4W x 259 Psychrobacter cryohalolentis K5 uid58373 x 260 Rhodobacter sphaeroides ATCC 17025 x uid58451 261 Rhodococcus erythropolis PR4 uid59019 x 262 Rhodopseudomonas palustris TIE 1 x uid58995 263 Roseburia inulinivorans DSM 16841 x 264 Roseobacter sp. MED193 x 265 Ruminococcaceae bacterium D16 x 266 Runella slithyformis DSM 19594 uid68317 x 267 Saccharophagus degradans 2 40 uid57921 x 268 Salmonella enterica serovar Typhimurium x 14028S uid86059 269 Salmonella enterica serovar Typhimurium x 798 uid158047 270 Salmonella enterica serovar Typhimurium x LT2 uid57799 271 Salmonella enterica serovar Typhimurium x SL1344 uid86645 272 Salmonella enterica serovar Typhimurium x ST1660 06 uid190371 273 Salmonella enterica serovar Typhimurium x ST4 74 uid84393 274 Salmonella enterica serovar Typhimurium x STm1 uid181283 275 Salmonella enterica serovar Typhimurium x STm12 uid181362 276 Salmonella enterica serovar Typhimurium x STm2 uid181284 277 Salmonella enterica serovar Typhimurium x STm3 uid181357 278 Salmonella enterica serovar Typhimurium x STm4 uid181358 279 Salmonella enterica serovar Typhimurium x STm8 uid181355 280 Salmonella enterica serovar Typhimurium x STm9 uid181356 281 Salmonella enterica serovar Typhimurium x T000240 uid84397 282 Salmonella enterica serovar Typhimurium x uid86061 283 Salmonella enterica serovar Typhimurium x UK 1 uid87049 284 Salmonella enterica subsp. enterica serovar x 4,[5],12:i:- str. CVM23701 285 Salmonella enterica subsp. enterica serovar x Enteritidi 286 Salmonella enterica subsp. enterica serovar x Infanti 287 Salmonella enterica subsp. enterica serovar x Saintpau 288 Salmonella enterica subsp. enterica serovar x Typhimuriu 289 Salmonella enterica subsp. enterica serovar x Uganda str 290 Selenomonas sputigena ATCC 35185 x 291 Selenomonas sputigena ATCC 35185 x uid55329 292 Shewanella ANA 3 uid58347 x 293 Shewanella baltica OS625 x 294 Shewanella MR 4 uid58345 x 295 Sinorhizobium meliloti CCNWSX0020 x 296 Slackia heliotrinireducens DSM 20476 x uid59051 297 Sphingobium indicum B90A x 298 Sphingobium yanoikuyae XLDN2-5 x 299 Spirosoma linguale DSM 74 uid43413 x 300 Sporolactobacillus vineae DSM 21990 = x SL153 301 Sporosarcina newyorkensis 2681 x 302 Streptomyces sp. SPB78 x 303 Sulfuricurvum kujiense DSM 16994 x uid60789 304 Synechococcus PCC 6312 uid182934 x 305 Syntrophus aciditrophicus SB uid58539 x 306 Thauera MZ1T uid58987 x 307 Thermacetogenium phaeum DSM 12270 x uid177811 308 Thermoanaerobacterium x thermosaccharolyticum DSM 571 uid51639 309 Thiocystis violascens DSM 198 uid74025 x 310 Thioflavicoccus mobilis 8321 uid184343 x 311 Thiothrix nivea DSM 5205 x 312 Vibrio cholera CIRS 101 x 313 Vibrio cholerae 4260B x 314 Vibrio cholerae CP1035(8) x 315 Vibrio cholerae CP1048(21) x 316 Vibrio cholerae CP1050(23) x 317 Vibrio cholerae H1 x 318 Vibrio cholerae HC-17A1 x 319 Vibrio cholerae HC-17A2 x 320 Vibrio cholerae HC-19A1 x 321 Vibrio cholerae HC-22A1 x 322 Vibrio cholerae HC-23A1 x 323 Vibrio cholerae HC-28A1 x 324 Vibrio cholerae HC-32A1 x 325 Vibrio cholerae HC-37A1 x 326 Vibrio cholerae HC-38A1 x 327 Vibrio cholerae HC-39A1 x 328 Vibrio cholerae HC-40A1 x 329 Vibrio cholerae HC-41A1 x 330 Vibrio cholerae HC-43A1 x 331 Vibrio cholerae HC-46A1 x 332 Vibrio cholerae HC-47A1 x 333 Vibrio cholerae HC-48A1 x 334 Vibrio cholerae HC-48B2 x 335 Vibrio cholerae HC-49A2 x 336 Vibrio cholerae HC-55B2 x 337 Vibrio cholerae HC-56A2 x 338 Vibrio cholerae HC-57A2 x 339 Vibrio cholerae HC-60A1 x 340 Vibrio cholerae HC-61A1 x 341 Vibrio cholerae HC-61A2 x 342 Vibrio cholerae HC-62A1 x 343 Vibrio cholerae HC-62B1 x 344 Vibrio cholerae HC-64A1 x 345 Vibrio cholerae HC-65A1 x 346 Vibrio cholerae HC-67A1 x 347 Vibrio cholerae HC-68A1 x 348 Vibrio cholerae HC-69A1 x 349 Vibrio cholerae HC-70A1 x 350 Vibrio cholerae HC-71A1 x 351 Vibrio cholerae HC-72A2 x 352 Vibrio cholerae HC-77A1 x 353 Vibrio cholerae HC-7A1 x 354 Vibrio cholerae HC-80A1 x 355 Vibrio cholerae HC-81A1 x 356 Vibrio cholerae HC-81A2 x 357 Vibrio cholerae HCUF01 x 358 Vibrio cholerae HFU-02 x 359 Vibrio cholerae MJ 1236 uid59387 x 360 Vibrio cholerae MO10 x 361 Vibrio cholerae O1 2010EL 1786 uid78933 x 362 Vibrio cholerae O1 str. 2010EL-1792 x 363 Vibrio cholerae O1 str. 2010EL-1798 x 364 Vibrio cholerae O1 str. EC-0009 x 365 Vibrio cholerae O1 str. EC-0012 x 366 Vibrio cholerae O1 str. EC-0027 x 367 Vibrio cholerae O1 str. EDC-020 x 368 Vibrio cholerae O1 str. EM-1546 x 369 Vibrio cholerae O1 str. Inaba G4222 x 370 Vibrio cholerae O1 str. Nep-21106 x 371 Vibrio cholerae O1 str. Nep-21113 x 372 Vibrio cholerae O1 str. NHCC-004A x 373 Vibrio cholerae O1 str. NHCC-006C x 374 Vibrio cholerae O1 str. NHCC-010F x 375 Vibrio cholerae O1 str. PCS-023 x 376 Vibrio cholerae VC4370 x 377 Vibrio harveyi CAIM 1792 x 378 Vibrio shilonii AK1 x 379 Vibrio tubiashii ATCC 19109 x 380 Vibrio cholerae HC-20A2 x 381 Vibrio cholerae HC-21A1 x 382 Vibrio cholerae HC-42A1 x 383 Vibrio cholerae HC-51A1 x 384 Vibrio cholerae O1 str. 3582-05 x 385 Xanthomonas vesicatoria ATCC 35937 x 386 Yersinia ruckeri ATCC 29473 x 387 Zymomonas mobilis NCIMB 11163 x uid41019 388 Clostridium clariflavum DSM 19732 xx Genome contains two BREX uid82345 systems of type #1 389 Clostridium saccharolyticum WM1 xx Genome contains two BREX uid51419 systems of type #1 390 Fusobacterium necrophorum subsp xx Genome contains two BREX systems of type #1 391 Gallionella capsiferriformans ES 2 uid51505 xx Genome contains two BREX systems of type #1 392 Magnetospirillum sp. SO-1 xx Genome contains two BREX systems of type #1 393 Methanoplanu xx Genome contains two BREX systems of type #1 394 Methanospirillum hungatei JF 1 uid58181 xx Genome contains two BREX systems of type #1 395 Salmonella enterica serovar Typhimurium xx Genome contains two BREX STm11 uid181361 systems of type #1 396 Stomatobaculum longum (Lachnospiraceae xx Genome contains two BREX bacterium ACC2) systems of type #1 397 Syntrophomonas wolfei Goettingen xx Genome contains two BREX uid58179 systems of type #1 398 Vibrio cholerae B33 xx Genome contains two BREX systems of type #1 399 Haloarcula argentinensis DSM 12282 x 400 Haloarcula hispanica ATCC 33960 x uid72475 401 Halobacterium salinarum R1 uid61571 x 402 halophilic archaeon DL31 uid72619 x 403 Halopiger xanaduensis SH6 uid68105 x 404 Halorhabdus utahensis DSM 12940 x uid59189 405 Halorubrumlacus profundi ATCC 49239 x uid58807 406 Halosimplex carlsbadense 2-9-1 x 407 Natrinema pellirubrum DSM 15624 x uid74437 408 Natronorubrum tibetense GA33 x 409 Anaeromyxobacter dehalogenans 2CP 1 x uid58989 410 Planctomyces limnophilus DSM 3776 x uid48643 411 Rhodopirellula sp. SWK7 x 412 Haliangium ochraceum DSM 14365 xx x Genome contains two BREX uid41425 systems of type #6 and one ot type #2 413 Nitrococcus mobilis Nb-231 x x Genome contains two BREX systems: #3 and #2 414 Anaerobaculum mobile DSM 13181 x x Genome contains two BREX uid168323 systems: #3 and #4 415 Acetohalobium arabaticum DSM 5501 x uid51423 416 Acidothermus cellulolyticus 11B uid58501 x 417 Alcanivorax hongdengensis A-11-3 x 418 Bacillus cereus 03BB108 x 419 Bacillus cereus BAG2X1-1 x 420 Bacillus methanolicus MGA3 x 421 Bacillus cereus W x 422 Bacteroides vulgatus ATCC 8482 uid58253 x 423 Caldicellulosiruptor kristjanssonii 177R1B x uid60393 424 Caloramator australicus RC3]Length = 15 x 425 Chloroflexus aggregans DSM 9485 x uid58621 426 Clostridium papyrosolvens DSM 2782 x 427 Clostridium thermocellum YS x 428 Desulfotomaculum nigrificans DSM 574 x 429 Desulfovibrio aespoeensis Aspo 2 uid42613 x 430 Dethiosulfovibrio peptidovorans DSM x 11002 431 Dichelobacter nodosus VCS1703A x uid57643 432 Ectothiorhodospir x 433 Enterococcus faecium 1,231,501 x 434 Kingella denitrificans ATCC 33394 x 435 Lachnospiraceae bacteriu x 436 Methanocaldococcus FS406 22 uid42499 x 437 Methanosalsum zhilinae DSM 4017 x uid68249 438 Methylacidiphilum infernorum V4 uid59161 x 439 Methyloversatilis universalis FAM5 x 440 Nitrosococcus oceani AFC27 x 441 Nitrosococcus oceani ATCC 19707 x uid58403 442 Nitrosococcus watsonii C 113 uid50331 x 443 Pelotomaculum thermopropionicum SI x uid58877 444 Planctomyces brasiliensis DSM 5305 x uid60583 445 planctomycete KSU-1 x 446 Pseudoalteromonas marina mano4 x 447 Pseudomonas sp. GM55 x 448 Syntrophothermus lipocalidus DSM 12680 x uid49527 449 Thermanaerovibrio acidaminovorans DSM x 6589 uid41925 450 Thermoanaerobacter brockii finnii Ako 1 x uid55639 451 Thermoanaerobacter ethanolicus CCSD1 x 452 Thermoanaerobacter ethanolicus JW 200 x 453 Thermoanaerobacter italicus Ab9 uid46241 x 454 Thermoanaerobacter pseudethanolicus x ATCC 33223 uid58339 455 Thermoanaerobacterium saccharolyticum x JW SL YS485 uid167781 456 Thermoanaerobacterium x thermosaccharolyticum M0795 uid184821 457 Thermoanaerobacterium xylanolyticum LX x 11 uid63163 458 Thermoplasmatales archaeon SCGC AB- x 539-N05 459 Treponema primitia ZAS-1 x 460 Vibrio scophthalmi LMG 19158 x 461 Parvibaculum lavamentivorans DS 1 xx Genome contains two BREX uid58739 systems of type #3 462 Amycolatopsis azurea DSM 43854 x 463 Bradyrhizobium sp. ORS 375 x 464 Burkholderia thailandensis E264 x 465 Burkholderia thailandensis E264 uid58081 x 466 Candidatus Accumulibacter phosphatis x clade IIA UW 1 uid59207 467 Corallococcus coralloides DSM 2259 x uid157997 468 Corynebacterium variabile DSM 44702 x uid62003 469 Dietzia cinnamea P4 x 470 Frankia CcI3 uid58397 x 471 Frankia EuI1c uid42615 x 472 Frankia sp. EUN1f x 473 Gemmata obscuriglobus UQM 2246 x 474 Gordonia amicalis NBRC 100051 = JCM x 11271 475 Gordonia polyisoprenivorans NBRC 16320 x 476 Hahella chejuensis KCTC 2396 uid58483 x 477 Micromonospora aurantiaca ATCC 27029 x uid42501 478 Micromonospora sp. ATCC 39149 x 479 Mycobacterium gilvum PYR GCK x uid59421 480 Mycobacterium xenopi RIVM700367 x 481 Mycobacterium intracellulare ATCC 13950 x 482 Nocardia cyriacigeorgica GUH 2 uid89395 x 483 Phaeospirillum molischianum DSM 120 x 484 Planctomyces maris DSM 8797 x 485 Polaromonas naphthalenivorans CJ2 x uid58273 486 Pseudomonas stutzeri NF13 x 487 Rhodococcus triatomae BKS 15-14 x 488 Rhodococcus ruber BKS 20-38 x 489 Saccharomonospora cyanea NA-134 x 490 Saccharomonospora glauca K62 x 491 Saccharomonospora viridis DSM 43017 x uid59055 492 Saccharomonospora xinjiangensis XJ-54 x 493 Saccharopolyspora erythraea NRRL 2338 x 494 Saccharopolyspora erythraea NRRL 2338 x uid62947 495 Saccharothrix espanaensis DSM 44229 x uid184826 496 Singulisphaera acidiphila DSM 18658 x uid81777 497 Sorangium cellulosum So ce 56 uid61629 x 498 Streptomyces coelicolor A3 2 uid57801 x 499 Streptomyces griseus NBRC 13350 x uid58983 500 Streptomyces turgidiscabies Car8 x 501 Streptomyces gancidicus BKS 13-15 x 502 Thermobifida fusca YX uid57703 x 503 Thermobispora bispora DSM 43833 x uid48999 504 Aciduliprofundum MAR08 339 uid184407 x 505 Candidatus Desulforudis audaxviator x MP104C uid59067 506 Coprothermobacter proteolyticus DSM 5265 x uid59253 507 Cyanobacterium stanieri PCC 7202 x uid183337 508 Denitrovibrio acetiphilus DSM 12809 x uid46657 509 Desulfitobacterium dichloroeliminans LMG x P 21439 uid82555 510 Geobacter M21 uid59037 x 511 Prevotella denticola F0289 uid65091 x 512 Thermomicrobium roseum DSM 5159 x uid59341 513 Thermotoga petrophila RKU 1 uid58655 x 514 Thioalkalivibrio sp. K90mix x

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”, and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”. John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. No. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames. B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames. B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Materials and Methods

Genomic Data and Molecular Phylogeny of the pglZ Protein—

A set of 1447 completely sequenced prokaryotic genomes (1336 bacterial and 111 archaeal genomes) were downloaded from the NCBI FTP site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) and used for subsequent analyses. Several pglZ protein sequences were used as a query in a PSI-BLAST search against the 1447 prokaryotic genomes with an inclusion threshold e-value of 0.001. Proteins that did not contain the pglZ domain or that were <600 amino acids length were filtered out. The remaining protein sequences were used to build a pglZ tree as follows: Amino acid sequences were aligned using the MAFFT algorithm [Katoh et al. Nucleic acids research (2002) 30: 3059-3066]. The Fourier transform approximation was disabled, and substitution rates were modeled with JTT [Jones et al. Computer applications in the biosciences: CABIOS (1992) 8: 275-282] and BLOSUM45 matrix, which is suitable for diverged sequences. The gene tree was reconstructed using the probabilistic RA×ML algorithm, with 100 bootstrap replicates, substitutions modeled with JTT (Jones et al. 1992), while allowing for rate variability among sites. For simplicity, the tree presented in FIG. 1A shows only the 115 pglZ protein sequences that are part of a complete Bacteriophage Exclusion (BREX) system. The brxC/pglY phylogeny tree was built in the same manner.

Identification of Bacteriophage Exclusion (BREX) Types 1-6—

System types were characterized based on manual observation of phyletic clusters in the pglZ tree. The specific genes associated with each pglZ phyletic type were defined using the IMG genome browser (www://img.jgi.doe.gov/cgi-bin/w/main.cgi). A representative protein sequence of each of the individual genes (Table 1 below) was then used as query in a PSI-BLAST search with an inclusion threshold e-value of 0.05. Only gene clusters containing the two core genes (pglz and brxC/pglY) and at least two additional genes were considered, under the added constraint that the genomic distance between the first and last genes in the system be under 30 kb. In the case of pglY, homology was based on the shared motifs (the p-loop motif GXXXXGK(T/S) (DUF2791, SEQ ID NO: 6162) and DUF499 combined with the conserved size of the gene in the different subtypes (˜1200 amino acids). The filtered clusters were manually assigned to systems according to gene content. Only clusters containing the complete set of genes or missing one non-core gene were included in the final set (Tables 2-7 below). In the case of BREX type 2, systems missing both brxD and brxHI were also included in the final set. The blastx program was used to scan intergenic regions in the clusters for unnanotated genes. Protein domains were annotated using the conserved domain database (CDD)³⁴ and HHpred³⁵. In the latter case, queries were carried out using representative sequences against the PDB, SCOP, interpro, pfam, smart, tigrfam and COG databases using default search parameters. The blastx program was used to scan intergenic regions in the clusters for un-annotated genes.

The consensus organisms tree (represented in FIG. 4) was derived from the NCBI “common tree” downloaded from the NCBI Taxonomy portal. In order to check whether subtypes were overrepresented in specific bacterial phyla the two following ratios were compared, using a hypergeometric statistical test: (i) Number of instances of a specific subtype in a specific phylum/total number of the specific subtype in bacteria; (ii) Total number of genomes of the specific phylum analyzed/total number of bacterial genomes analyzed. P-value ≦0.05 was considered statistically significant following Benjamini and Hochberg correction for multiple testing.

Extensive Identification of BREX Systems in Prokaryotic Genomes

A set of 2263 completely sequenced prokaryotic genomes and 5493 draft genomes was downloaded from the NCBI FTP site (ftp://ftp.ncbi.nih.gov/genomes/Bacterial and ftp://ftp.ncbi.nih.gov/genomes/Bacteria_DRAFT/, respectively) and used for subsequent analyses. A representative protein sequence of each of the 13 genes (Table 1 below) was then used as query in a PSI-BLAST search against the 7756 completely sequences and draft genomes with an inclusion threshold e-value of 0.05. Only gene clusters containing the two core genes (pglZ and brxC/pglY) and at least two additional genes were considered and listed in tables 10-15 below.

Strain Construction—

The type 1 BREX system was amplified in fragments from the Bacillus cereus H3081.97 genome from position 89,288-103,514 (GenBank ABDL02000007.1, SEQ ID NO: 6164). The PCR-amplified fragments were assembled to a circular plasmid in S. cerevisiae using the pYES1L vector (Invitrogen), transformed into Eccherichia coli BL21 AI and amplified, and then integrated into the proB gene in Bacillus subtilis BEST7003, along with a chloramphenicol resistance cassette. The DNA sequence of the plasmid used for the integration is depicted in SEQ ID NO: 6139. The primers used for construction are depicted in SEQ ID NO: 6140-6151. The presence of the intact BREX system within Basillus subtilis BEST7003 was confirmed by PCR and Illumina-based whole genome sequencing. Primers sequences are depicted in SEQ ID NO: 6152-6161. Control strains contain only the chloramphenicol resistance cassette integrated at the proB locus. The pglX deletion strain was constructed in a similar manner with PCR fragments that created a deletion from position 94,655-98,163 (GenBank ABDLO2000007.1, SEQ ID NO: 6164), leaving only 31 nucleotides of the pglX gene. The DNA sequence of the plasmid used for the integration is depicted in SEQ ID NO: 6210.

Growth Dynamics of Phage Infected Cultures—

Overnight cultures were diluted 1:100 in LB media supplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂ and then grown to an OD₆₀₀ of 0.06 in a 96-well plate format. Phages were added at a multiplicity of infection (MOI) ranging from 10⁻³ to 10⁻⁴. High concentration phage infections were performed at MOI ranging from 0.05 to 5. Optical density measurements at a wavelength of 600 nm were taken every 13 minutes using a TECAN infinite 2000 plate reader.

Plaque Assays—

Small drop plaque assays were initially performed using 0.75% agar plates containing bacterial cultures that were diluted 1:13 in LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl₂. Serial dilutions of the phage between 2×10⁰ and 2×10⁵ plaque-forming units (pfu) were spotted on these plates and plaques were counted after overnight growth at room temperature. Further confirmation of plaque numbers was performed by an agar overlay assay. The bottom agar was composed of LB media supplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂ and 1.5% agar. The top agar was prepared by diluting overnight bacterial cultures 1:30 in LB media supplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂ and 0.5% agar with the addition of serial dilutions of the phage. Plaques were counted after overnight growth at room temperature.

One-Step Phage Growth Curve Assays—

One-step phage growth curve experiments were performed as described by Carlson [E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: biology and applications, CRC Press, Boca Raton. Fla. Appendix p. 437-494]. Logarithmic phase cultures were infected with either phage SPO1 or φ3T at an MOI of 0.05. Following 18 minutes of growth at 37° C. the infected culture was diluted 1:10,000, to reduce the likelihood of phage infection following cell lysis. To evaluate the number of infective centers and extracellular phage present in the infected culture, samples were taken at specific time points throughout the incubation period, mixed with a phage-sensitive Basillus subtilis strain and plated using the agar overlay method described hereinabove. Phage adsorption was inferred by evaluating the number of extracellular phage present in the mixture 15 minutes following infection. This was assayed by mixing the infection mixture with chloroform, incubating it at 37° C. for 4 minutes, followed by incubation for 4 minutes on ice, and 30 minutes at room temperature. The aqueous phase was then mixed with a phage-sensitive Basillus subtilis strain and plated using the agar overlay method described hereinabove. The addition of chloroform leads to bacteria killing, including phage adsorbed bacteria. At early time points, phage have not yet assembled inside the cell, and are therefore unable to form plaques. Thus, the derived results allow evaluation of the extracellular phage levels. A drop in extracellular phage levels indicates that adsorption has occurred.

DNA Extraction—

DNA extraction was performed by suspending cells in 50 mM EDTA pH 8.0 with a lytic enzyme (lysozyme. Sigma) for 90 minutes at 37° C. followed by centrifugation for 2 minutes at 13,000 g and removal of supernantat. The cells were then lysed by adding a Nuclei Lysis Solution (Promega, cat no. A7941) for 5 minutes at 80° C. followed by addition of Rnase A (10 mg/ml) for 30-60 minutes at 37° C. The protein fraction was precipitated by adding 200 μl Protein Precipitation Solution (Promega, cat no. A795A), incubating the suspension for 5 minutes on ice followed by centrifugation at 13,000-16,000 g for 3 minutes. The supernatant was then transferred to a clean tube containing 600 μl isopropanol, mixed and centrifuged at 13,000-16,000 g for 3 minutes. The supernatant was removed and 600 μl 70% Ethanol was added to the pellet mixed and centrifuged at 13,000-16,000 g for 3 minutes. The ethanol was then aspirated and the pellet was air dried for a couple of minutes followed by resuspension in Qiagen Elution buffer.

Phage Infection Time Courses, Genomic DNA Sequencing and Methylation Analysis—

Phage infection time course cultures for both methylome analysis, detection of lysogeny and relative phage abundance were performed at an MOI of 4. Phage infection time course cultures are practically cultures infected by phage and analyzed at specific time points (e.g. 0, 5, 10, 15, 20, 30 and 40 minutes following infection). Uninfected cultures analyzed at the same time points served as control. Cell pellets were washed three times in 10 mM Tris pH 7.4 to remove unadsorbed phage, followed by DNA extraction as described hereinabove. DNA library preparations and sequencing for methylome analysis were performed at the Yale Center for Genome Analysis (see Murray I A et al. (2012) Nucleic acids research 40: 11450-11462). To determine the relative abundance of bacterial and phage φ3T DNA levels, DNA was first fragmented using NEBNext® dsDNA Fragmentase (New England Biolabs Ibc.) according to manufacturer's instructions, followed by Illumina sequencing of the DNA libraries of φ3T phage-infected time course cultures. The sequences were mapped to the phage and host genomes as previously described [Wurtzel et al. PloS one (2010) 5: e15628]. Sequences shared by both Basillus subtilis BEST7003 and phage φ3T DNA were discarded from the dataset. The remaining mapped sequences were enumerated at each time point to compare the number of sequences mapped to the Basillus subtilis BEST7003 DNA relative to phage φ3T DNA and normalized to the genome size.

Detection of Phage Lysogeny—

Genomic DNA sequencing of a lysogen containing phage φ3T was performed using Illumina sequencing to determine the DNA sequence of the φ3T phage and the site of phage integration in the genome. The integration of the φ3T phage was determined at a GTAGG site on the Basillus subtilis BEST7003 bacterial genome at position 2106060-2106064. Multiplex PCR assays were used to detect phage φ3T DNA, Basillus subtilis BEST7003 DNA, and the novel junction created in the lysogenized strain. Primers used to detect phage φ3T were GAGGTTCGCTACGGGCGAAAT (SEQ ID NO: 6211) and TCTCTGCTTGATITCGTCCATGA (SEQ ID NO: 6212). Primers for detection of Basillus subtilis BEST7003 and the unique junction found in the lysogen were TGCCTGCATGAGCTGATITG (SEQ ID NO: 6213) and GCAGGAATGAATGGTGGATATTG (SEQ ID NO: 6214); and TCATGCTCCGGATTTGCGAT (SEQ ID NO: 6215) and TGCCTCCITTCGATITTGTTACC (SEQ ID NO: 6216), respectively.

Structural Homology Between brxA and NusB—

Alignment between brxA from Magnetospirillum magneticum (PDB entry 3BHW) and NusB from Aquifex aelicus (PDB entry 3R2C) was performed using the MultiProt web server and presented using PyMol (Schrödinger, Inc, Portland, Oreg., USA).

Agarose Gel and Southern Blot Analysis—

200 ng of undigested genomic DNA was run on a TAE agarose gel. The agarose gel was depurinated in 0.25 N HCl for 20 minutes, rinsed in ddH₂O, and soaked in denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 10 minutes. The DNA was then transferred onto HybondXL membrane (Amersham) by capillary transfer in denaturation buffer and the membranes were baked for 2 hours at 80° C. DNA for probes was labeled with ∝³²P-dCTP using the High Prime Kit (Roche Cat no. 11 585 584 001) according to manufacturer's instructions. Phage φ3T specific primers were PTG111: TGGATTTCAGCTGGGGAAGA (SEQ ID NO: 6217) and PTG112: AACTTGTCTCTATCTTATCACCTGT (SEQ ID NO: 6218). The membranes were incubated overnight with the probe at 65° C. in hybridization buffer (7% SDS, 0.5 M NaPhosphate pH 7.2, 10 mM EDTA), washed twice with 2×SSC, 0.1% w/v SDS, washed twice with 1×SSC, 0.1% w/v SDS, then four times with 0.2×SSC, 0.1% w/v SDS and exposed to phosphorimager screen and visualized.

RNA sequencing and 5′ and 3′ RACE—were performed as described in Wurtzel O. et al. (2012) Molecular Systems Biology, 8:583.

Example 1 Bacteriophage Exclusion (BREX) System is Abundant in Bacteria and Archaea

Previous reports demonstrated that various combinations of genes belonging to the Phage Growth Limitation (PGL) system, and predominantly pglZ, were enriched within ‘defense islands’ of bacteria and archaea^(9,13). The present inventors have initially performed homology searches in 1447 bacterial and archaeal genomes in order to understand whether there is higher order organization amongst pglZ and its associated genes. These homology searches found 144 occurrences of pglZ amongst the 1447 bacterial and archaeal genomes analyzed. Phylogenetic tree reconstruction of these pglZ proteins showed clear clustering of pglZ into several defined phyletic groups (FIG. 1A). By analyzing the genomic context of pglZ in each of these groups, a distinct set of 13 genes strongly associated with pglZ was identified (Table 1 below). The composition and order of these genes were highly coherent within each phyletic group but differed between the clades, defining clear organizational subtypes, with each subtype composed of 4-8 genes. Of the 14 genes, only brxC/pglY and pglZ recurred in all system subtypes, with the additional genes being subtype-specific.

The present inventors termed this overall system as ‘BREX’ (Bacteriophage Exclusion, previously termed PYZA), and defined six major BREX types according to the phylogeny and operon organization (FIG. 1A). Thus, overall 135 BREX systems were found in 9% (126/1447) of all genomes analyzed, usually appearing on the chromosomal DNA (Tables 2-8 above). BREX type 1, the most common form of BREX, appeared 79 times in 75 genomes (Table 8 below), and is typically composed of 6 genes arranged in a conserved order (FIG. 1A).

Taken together pan genomic analysis revealed a novel broadly distributed multi-gene system which the present inventors denoted BREX system. This family of systems exists in almost 10% of sequenced microbial genomes, and can be divided into six coherent subtypes in which the gene composition and order is conserved (for further details see Example 2 below). Each BREX subtype contains 4-8 genes. By definition, all BREX subtypes contain a pglZ-domain gene. In addition, all of them harbor a large protein with a P-loop motif. The P-loop motif (GXXXXGK[T/S]) is a conserved ATP/GTP binding motif that is ubiquitously found in many ATP-utilizing proteins such as kinases, helicases, motor proteins and proteins with multiple other functions [Thomsen and Berger Molecular microbiology (2008) 69: 1071-1090]. In general, the P-loop containing genes in the various BREX subtypes share little homology: for example, the brxC gene of BREX type 1 and pglY gene of BREX type 2 share homology only across 4% of their protein sequence, and this homology is concentrated around the P-loop motif (FIG. 2). Despite the low homology, distant homology analysis with HHpred [Soding Bioinformatics (2005) 21: 951-960] showed that they share a domain denoted DUF499 (Table 1). It is therefore suggested that the P-loop containing genes in all six BREX subtypes share a similar role in the system, and hence these genes are denoted herein as brxC/pglY and referred to as having a common function (Table 1). Apart from the two core genes pglZ and brxClpglY that appear in each of the six BREX subtypes, the remaining genes are subtype-specific or restricted to only a subset of the BREX subtypes.

TABLE 1 Genes composing the BREX systems Subsystems in which gene Median gene Gene appears Associated domains Domain annotation size (aa) pglZ Core gene pfam08665 Alkaline phosphatase 835 brxC/ Core gene DUF499, ATP binding 1208 pglY DUF2791 (pfam10923) brxA 1, 3, 5, 6 DUF1819 Unknown function 232 (pfam08849) pglX 1, 2, 5, 6 Pfam13659 Adenine-specific 1175 (COG1002/COG0286) methylase brxL 1, 4 COG4930 Lon-like protease 682 brxHII 3, 5 COG0553 DNA/RNA helicases 965 brxHI 2, 6 COG1201 Lhr-like Helicase 712 brxD 2, 6 DUF2791 ATP binding 442 (pfam10923) brxE 6 Unknown function 201 brxB 1, 5, 6 DUF1788 Unknown function 193 (pfam08747) pglXI 3 COG0863/COG1743 Adenine-specific 920 (pfam01555) methylase brxF 3 ATPase 158 pglW 2 COG0515 Serine/threonine protein 1413 kinase brxP 4 COG0175 Phosphoadenosine 774 (pfam01507), phosphosulfate, reductase pfam13182

Example 2 Characterization of the Six BREX Types

Six types of BREX system were characterized based on manual observation of phyletic clusters in the pglZ tree (FIGS. 1A-B and Tables 2-8 above).

Type 1 BREX—

The most common BREX system identified comprises a 6-gene cluster arranged in a highly conserved order in a diverse array of bacteria and archaea (FIG. 1A-C and Table 2 above). Two of the six genes found in this conserved cluster share homology with genes from the previously reported Pgl system¹¹: pglZ, coding for a protein with a predicted alkaline phosphatase domain, and pglX, coding for a protein with a putative methylase domain. The four additional genes include (i) a Ion-like protease-domain gene, denoted herein as brxL; (ii) a gene, denoted herein as brxA; (iii) a gene, denoted herein as brxB; and (iv) a large, ˜1200 amino acid protein with an ATP binding motif (GXXXXGK[T/S]), denoted herein as brxC. Although this does not resemble any classical combination of genes currently known to be involved in phage defense, the preferential localization of this conserved gene cluster in the genomic vicinity of other defense genes suggests that it could form a novel phage defense system.

The brxA family of proteins are, on average, 232 amino acids long and do not share sequence similarity with any domain of known function. However, as part of the protein structure initiative the structure of the type 1 brxA protein from Magnetospirillum sp. SO-1 was solved (PDB entry 3BHW). A significant structural similarity, spanning 44 amino acids of the brxA protein, was found between the Magnetospirillum brxA and the 148 amino acids RNA binding protein NusB (PDB entry 3R2C)[Stagno et al. Nucleic acids research (2011) 39, 7803-7815]. NusB is part of an anti-termination complex that enables proper ribosomal RNA transcription in E. coli. The anti-termination complex is initiated by binding of NusB and NusE to a BOXA site, a specific sequence on the nascent rRNA. The complex, which assembles additional proteins such as NusE, NusG and NusA, modifies RNA polymerase to enable readthrough past Rho-dependent transcriptional terminators that are present in the rRNA sequence [Luttgen et al. Journal of molecular biology (2002) 316, 875-885]. NusB was also shown to be essential for the life cycle of bacteriophage λ, and specifically for the transition from early transcription into late transcription. In the middle stages of infection, the phage N protein couples with NusB. NusE, NusA and NusG to direct the host RNA polymerase to read through the terminators of the phage immediate early genes and proceed to transcription of middle genes [Stagno et al. Nucleic acids research (2011) 39, 7803-7815]. As demonstrated in FIG. 3, the brxA protein displays significant structural homology to NusB. This similarity spans the RNA-binding interface, as well as part of the protein:protein interaction interface with NusE. In light of this similarity, it is proposed that brxA is also an RNA binding protein. It is further speculated that this protein has a role in interfering with the phage infection cycle by disrupting anti-termination events essential for the phage cycle.

Type 2 BREX—

Type 2 BREX system encloses the phage defense system originally described as PGL¹⁰ (FIGS. 1A-B and Table 6 above). However, while the described PGL system composed four genes (pglW, X, Y and Z)¹¹, in 89% of the cases (16/18 instances) two additional genes, denoted herein as brxD and brxHI, were found to be associated with the system. Given that both these genes appear in the same order in the type 6 BREX system (FIG. 1A), it is suggested that these genes play an integral part of the type 2 system. The first gene, brxD, encodes a small protein predicted to bind ATP, while the second gene, brxHI encodes a predicted helicase. In addition, the serine-threonine kinase (pglW) exists exclusively in this subtype.

Type 3 BREX—

The type 3 BREX system was observed in 20 of the genomes analyzed (FIGS. 1A-B and Table 5 above). Both systems type 1 and type 3 contain the short protein denoted herein as brxA, which has no known function. In addition, both type 1 and type 3 systems contain a gene encoding an adenine-specific DNA methylase (pglX and pglXI for subtypes 1 and 3, respectively), although the methylase domain differs between the subtypes (pfam13659 and pfam01555 in pglX and pglXI, respectively). It is therefore likely that pglX and pglxXI perform the same DNA methylation function although they do not share sequence homology. BREX type 3 systems contain a predicted helicase (denoted herein as brxHII) instead of the ion-like protease present in type 1. In addition, the brxB gene present in type 1 has been replaced with another protein, denoted herein as brxF.

Type 4 BREX—

The type 4 BREX is composed of four genes (FIGS. 1A-B and Table 7 above), two of which are the core brxC/pglY and pglZ genes, and the third is the lon-like protease (denoted herein as brxL). The fourth gene, denoted herein as brxP, is subtype-specific and contains a phosphoadenylyl-sulfate reductase domain (COG0175/pfam01507). This domain was previously associated with the phage-resistance DND system that performs sulfur modifications on the DNA backbone, providing an additional link between BREX systems and phage resistance¹⁶⁻¹⁸.

Type 5 BREX and Type 6 BREX—

The two least common BREX subtypes, type 5 and type 6, are similar to the type 1 BREX system but contain some additional variations (FIGS. 1A-B and Tables 3 and 4 above). In type 5 BREX, the Ion-like protease present in type 1 BREX has been replaced by a helicase-domain gene (denoted herein as brxHII) carrying a COG0553 domain, and brxC/pglY has been duplicated (FIG. 1A and Table 3 above). In subfamily 6, the protease present in type 1 has been replaced by two genes, a helicase with a COG1201 domain (brxHI), and an ATP/GTP binding protein (brxD) (FIG. 1A and Table 4 above); a pair which also appears in type 2 BREX. Type 6 BREX systems also contain an additional gene found as the first gene in the cluster, which was denoted herein as brxE.

Taken together, 135 of the 144 (94%) pglZ genes detected in microbial genomes were found to be embedded as part of one of the six BREX systems described (Table 8 above), and 7 of the remaining pglZ genes were clearly part of degraded (probably pseudogenized) systems. In most cases a single BREX system per organism was found, with only 8 (6.5%) of genomes harboring more than one subtype (Table 8 above). In addition, in 14% (19/135) of the identified systems, one of the genes was either missing or has become a pseudogene (tables 2-7 above), possibly representing inactivated systems. A similar tendency for gene loss was observed for the CRISPR-Cas system, and it was suggested that CRISPR-Cas inactivation is caused by fitness cost imposed by this defense system^(2,19,20). Phage defense systems often encode toxic genes²¹, and it is possible that such toxic genes encoded by BREX systems impose fitness cost and lead to gene loss in the absence of phage pressure.

Example 3 Type 1 BREX Confers Resistance to Phage Infection in Bacillus Subtilis

To determine whether the BREX system provides protection against phage infection, the complete type 1 BREX system from Bacillus cereus H3081.97 (FIG. 4A), composing the brxA, brxB, brxC, pglX, pglZ, and brxL genes was integrated into a Bacillus subtilis strain lacking an endogenous BREX system. The type 1 BREX system was integrated into the Bacillus subtilis BEST7003 genome, a derivative of Bacillus subtilis 168 that lacks the SPβ lysogenic phage, avoiding the potential for superinfection exclusion. Proper integration of the intact system was verified by PCR and complete genome sequencing. RNA-sequencing further verified that the genes of the integrated system are transcribed in Bacillus subtilis when grown in exponential phase in rich medium. Furthermore, using 5′ and 3′ RACE it was determined that the system is transcribed as two operons with the first four genes, brxA-brxB-brxC-pglX, forming a single transcriptional unit, while the last two genes, pglZ-brxL, are co-expressed as a second transcriptional unit (FIG. 4B). The observation that the genes in the putative BREX system are co-transcribed as two long polycistronic mRNAs further supports they work together as components of a functional system.

Ten Bacillus subtilis phages were selected for phage infection experiments, spanning a wide range of phage phylogeny, from T4-like Myoviridae (SPO1 and SP82G), lambda-like Siphoviridae (φ105, rho10, rho14 and SPO2) and SPβ-like Siphoviridae (Φ3T, SPβ, SP16 and Zeta). Two of the phages are obligatory lytic (SPO1 and SP82G), while the remaining are temperate (See Table 9 below). The sensitivity of Bacillus subtilis strains either lacking or containing the BREX type 1 system to infection by the different phages was evaluated using both optical density measurements in a 96-well plate format, and double agar overlay and plaque assays (Table 9 below).

Upon phage infection, the Bacillus subtilis strain containing the BREX system showed complete resistance to five of the eight temperate phages tested (FIGS. 4D, I-L and Table 9 below). Growth curves of BREX-containing bacteria infected with these phages were indistinguishable from the uninfected bacteria, while rapid declines in optical density measurements were observed for the control strain lacking the BREX system, indicating lysis of the infected cells (FIGS. 4D, I-L). These results confirm that BREX is a phage defense system that provides protection against a wide array of phages, both virulent and temperate ones. In contrast, phage resistance was not observed upon infection with phage Φ105 and its close relatives, rho10 and rho14. Similar kinetics of cell lysis was observed for strains either containing or lacking the BREX system (FIGS. 4E, 4H and 4M and Table 9 below). Considering that phage Φ105 is estimated to share high (83-97%) genome homology with rho10 and rho14¹⁴, the inability of the type 1 BREX system to protect against these three phages could indicate that this phage family has evolved strategies to counteract the BREX defense, as has been observed with other bacterial defense systems¹⁵.

To further evaluate the level of protection provided by the type 1 BREX system against the tested temperate phages, plaque assays using increasing dilutions of phage were performed. For five of the temperate phages, no plaques were observed when the type 1 BREX-containing strain was challenged even with the highest phage concentrations, indicating that the type 1BREX system provides at least a 10⁵ fold protection against cell lysis upon phage infection (Table 9 below). The plaque assays also confirmed that phage Φ105 and its relatives evade type 1 BREX defense, with similar efficiencies of plating and plaque morphology observed in both type 1 BREX-containing and wild-type control strains (Table 9 below).

TABLE 9 Type 1 BREX protection against phage infection Infection Efficiency blocked of by BREX Phage Genus Family Life cycle BREX? protection^(a) SPβ SPβ-like Siphoviridae Temperate Yes >10⁵ SP16 SPβ-like Siphoviridae Temperate Yes >10⁵ Zeta SPβ-like Siphoviridae Temperate Yes >10⁵ Φ3T SPβ-like Siphoviridae Temperate Yes >10⁵ SPO2 Lambda- Siphoviridae Temperate Yes >10⁵ like Φ105 Lambda- Siphoviridae Temperate No 1 like rho10 Lambda- Siphoviridae Temperate No 1 like rho14 Lambda- Siphoviridae Temperate No 1 like SPO1 SPO1-like Myoviridae Obligatory Yes 8 * 10² ± lytic 0.02 SP82G SPO1-like Myoviridae Obligatory Yes 1.8 * 10¹ ± lytic 0.08 ^(a)Protection efficiency was calculated as the ratio between the number of plaques formed on the BREX-lacking strain divided by the number of plaques formed on the BREX-containing strain with the same phage titer, using increasing titers. Standard deviation was calculated from a biological triplicate of the plaque experiment.

The type 1 BREX-containing Bacillus subtilis strain also displayed some protection from the lytic SPO1 and SP82G phages in liquid culture experiments. Growth curves of the strain containing the BREX type 1 system infected with either SPO1 or SP28G phages were similar to the uninfected strains when evaluated for up to 12 hours following infection, while complete lysis was observed in infected control strains lacking the BREX system (FIGS. 4F-G). Endpoint analysis using plaque assays revealed a 10¹ fold reduction in plaque numbers in type 1 BREX-containing strains for SPO1 and SP82G phages (Table 9 above). In addition, plaque sizes were reduced 1.5-2 fold in the type 1 BREX-containing strain, to 47% and 65% the diameter of those observed in the control strain lacking the type 1 BREX system for SPO1 and SP82G, respectively (data not shown). These results are consistent with the observation that incubation of the type 1 BREX-containing strain with the two lytic phages for extended periods of time (>20 hours) often resulted in an eventual culture decline occurring at apparently stochastic points in time (FIGS. 5A-B).

To gain further insight into the nature of the incomplete type 1 BREX defense against these lytic phages, a one-step phage growth curve assay [Carlson Bacteriophages, Biology and Applications (eds. E Kutter, A Sulakvelidze) (2005) pp. 437-494. CRC Press, Florida.] was performed with SPO1. Briefly, this experiment involves mixing SPO1-infected cells with a SPO1-sensitive B. subtilis cells and plating them together using an agar overlay method. Phage bursts from successful infections are visualized as a single plaque on a lawn from the SPO1-sensitive B. subtilis strain, enabling an evaluation of the number of phages that have adsorbed and completed a successful infection cycle. As demonstrated in FIG. 6, enumeration of plaques during the first 45 minutes of the time course infection indicated that the SPO1 phage was able to complete the lytic cycle only in 9%±4 of the initially infected cells (FIG. 6). A delay in kinetics of the phage cycle was also observed, with phage bursts observed 75 minutes and 105 minutes following infection of BREX-lacking and type 1 BREX-containing cells, respectively (FIG. 6).

Taken together, these results suggest that the type 1 BREX system provides significant protection from infection by the lytic phages SPO1 and SP82G.

Example 4 The Mechanism of Action of Type 1 BREX

Due to the homology of a subset of the genes in the BREX system to genes in the previously described Pgl system¹⁰, it was necessary to examine whether BREX also functions through the described Pgl mechanism. The Pgl phenotype observed in S. coelicolor A3 predicts that the Pgl system does not confer resistance to phage first cycle of infection. One-step phage growth curve assays were used to examine the first infection cycle of phage Φ3T in type 1 BREX-containing cells. As demonstrated in FIG. 7, while the control strains lacking the BREX system displayed phage burst sizes of 61.5±10.2 particles per infected cell, there was no production of Φ3T phage during infections of type 1 BREX-containing Bacillus subtilis strains under similar conditions. To exclude the possibility that productive phage infection could occur at later time points, experiments were extended to 120 minutes (corresponds to 3 infection cycles in control strains) in type 1 BREX-containing Bacillus subtilis strains. As shown in FIG. 7, plaques were not observed in the type 1 BREX-containing Bacillus subtilis strains even at later time points. These results demonstrate that unlike the S. coelicolor Pgl system, the type 1 BREX system confers resistance to phage first cycle of infection.

Previous experiments with the S. coelicolor Pgl system also demonstrated that although the Pgl defense system prevents continued propagation of the temperate phage ΦC31, it does not block lysogeny of the phage¹⁰. To determine whether BREX also permits lysogeny, phage Φ3T integration into the Bacillus subtilis genome during infection was examined using a PCR assay. In control Bacillus subtilis strains lacking BREX, lysogeny was first detected 10 minutes following phage infection (FIG. 8). However, no evidence for phage integration into the host genome was found in type 1 BREX-containing Bacillus subtilis strains. Evaluation of lysogeny in bacterial colonies that survived the phage infection also indicated that none of the surviving type 1 BREX-containing colonies were lysogens, while all surviving colonies tested in strains lacking the BREX system were lysogenic for phage Φ3T.

One of the common forms of phage defense is abortive infection (Abi), where infected cells commit “suicide” before phage progeny are produced, thus protecting the culture from phage propagation⁴. To test whether the type 1 BREX system acts via an Abi mechanism, the type 1 BREX-containing Bacillus subtilis strains were infected with increasing concentrations of Φ3T phage. Using high multiplicity of infection (MOI) where nearly all bacteria are infected in the first cycle, massive cell lysis should be observed in the culture in the case of Abi. The results demonstrated that even at an MOI>1, no significant growth arrest or culture decline was found in the liquid culture (FIG. 9), suggesting that the type 1 BREX is not an Abi system.

In the next step, BREX ability to prevent phage adsorption and phage DNA replication were evaluated. As illustrated in FIG. 10, adsorption assays showed that Φ3T efficiently adsorbs to both type 1 BREX-containing and BREX-lacking Bacillus subtilis strains, indicating that type 1 BREX does not block adsorption. In order to test phage DNA replication within infected cells, total cellular DNA (including chromosomal DNA and intracellular phage DNA) was extracted at successive time points following a high-MOI infection by Φ3T; and the extracted DNA was sequenced by Illumina sequencing. Since host DNA is not degraded following Φ3T infection (FIG. 18), mapping sequenced reads to the reference Bacillus subtilis and Φ3T genomes, allowed quantification of the number of Φ3T genome equivalents per infected cell was quantified at each time point. In control Bacillus subtilis strains lacking BREX, phage DNA replication began between 10 and 15 minutes following infection, and 30 minutes following infection, phage DNA levels were elevated 81-fold relative to that observed at the 10 minutes time point (FIG. 11). In contrast, no increase in phage DNA levels was observed in type 1 BREX-containing Bacillus subtilis strains (FIG. 11).

To further test whether BREX leads to cleavage or degradation of phage DNA, the integrity of phage DNA was examined using Southern blot analysis on total cellular DNA extracted from phage-infected cells at increasing time points following infection. This analysis showed extensive replication of phage DNA in control Bacillus subtilis strains lacking type 1 BREX and affirmed no phage DNA replication in Bacillus subtilis strains containing type 1 BREX (FIG. 12). However, as shown in FIG. 12, the phage DNA in type 1 BREX-containing Bacillus subtilis strains appeared intact with no signs of phage DNA cleavage or processive degradation.

These results indicate that phage DNA replication does not occur in type 1 BREX-containing cells, that type 1 BREX does not lead to the degradation of phage DNA and that this system exerts its function at the early stages of the infection cycle.

As type 1 BREX contain an m6A DNA adenine methylase (pglX), the present inventors have evaluated whether either bacterial or phage DNA are methylated in a BREX-dependent manner. To this end, the PacBio sequencing platform that directly detects m6A modifications in sequenced DNA [Murray et al. Nucleic acids research (2012) 40: 11450-11462] was used. As demonstrated in FIG. 13A, the PacBio platform clearly detected m6A methylation on the 5^(th) position of the non-palindromic hexamer TAGGAG in chromosomal DNA extracted from type 1 BREX-containing Bacillus subtilis strains. Thus, while nearly all TAGGAG motifs were methylated in type 1 BREX-containing Bacillus subtilis strains (FIG. 13B), no methylation on this motif was observed in the control Bacillus subtilis strains lacking the BREX system. These results indicate that type 1 BREX drives motif-specific methylation on the genomic DNA of the bacteria in which it resides.

To examine whether BREX also methylates the invading phage DNA, total cellular DNA (including chromosomal DNA and intracellular phage DNA) was extracted at 10 and 15 minutes following a high-MOI infection by Φ3T and analysed PacBio sequencing. The results affirmed that the TAGGAG motifs in the bacterial genome were methylated throughout the infection. However, there was no methylation on these motifs in the phage genome at the time points tested during infection (data not shown).

The presence of bacterial-specific methylation could suggest that the type 1 BREX system encodes some kind of restriction/modification activity, and that the methylation of TAGGAG motifs in the bacterial genome may serve to differentiate between self and non-self DNA. This suggests that deletion of the methylase gene, pglX, would be detrimental to the cell, as the genomic TAGGAG motifs will no longer be protected from the putative restriction activity of BREX. However, as can be seen in FIG. 14A, deletion of the pglX from the type 1 BREX system that was integrated into Bacillus subtilis was not toxic to the cells. Moreover, type 1 BREX-containing Bacillus subtilis strains having a deletion of pglX were sensitive to all phage tested (for example FIG. 14B demonstrating strain sensitivity to Φ3T). These results show that pglX is essential for type 1 BREX-mediated phage resistance, and also suggest that the BREX mechanism of action is not consistent with a simple restriction/modification activity.

Taken together, these results suggest that phage adsorption occurs in type 1 BREX-containing strains. This system does not display the Pgl phenotype, and hence probably functions through a novel mechanism different than that of the Pgl system. In addition, the system methylates the host chromosomal DNA at a specific motif, and that this methylation is likely to be essential for the system's activity.

Example 5 Extensive Horizontal Transfer of BREX Systems

An examination of the distribution of BREX systems across microbial species showed that these systems undergo extensive horizontal transfer (FIG. 15). First, the distribution of systems across the species tree is interrupted (resembling the way CRISPRs are distributed in bacterial and archaeal genomes²²). Second, the pglZ tree is not consistent with the species tree, and closely related species can accommodate distantly related pglZ and vice versa. Nevertheless, phylogenetic trees reconstructed from additional BREX genes generally recapitulate the structure of the pglZ tree, suggesting that genes within specific BREX systems co-evolve and are co-horizontally transferred (For example FIG. 16).

The individual clades demonstrated in FIG. 1A separate close to the root of the PglZ tree and the six defined BREX subtypes are widespread across the entire bacterial and archaeal tree of life (FIG. 15), thus suggesting that the separation between the systems occurred at an ancient point in the evolutionary history of bacteria and archaea. The relative abundance of BREX type 1, and its appearance on several clades on the pglZ tree (FIG. 1A), suggest that this subtype might be the ancestral form of BREX. Despite the extensive horizontal transfer observed for the BREX systems, some clades show enrichment in specific subtypes: subtype 1 is enriched in Deltaproteobacteria (p=0.001); subtype 2 (the PGL system) appears almost solely in Actinobacteria (p=4.8×10⁻⁹); and subtype 5 is exclusive to the archaeal class Halobacteria. The enrichment of specific subtypes within specific phyla might link the ancestry of these subtypes to the phyla in which they are enriched; alternatively, phylum-specific BREX subtypes might rely on additional, phylum-specific cellular mechanisms that are not directly encoded in the BREX genes, or provide defense against phages that predominantly attack the specific phyla.

Within the 1447 genomes, the relative frequency of BREX in archaea (10%) was similar to that observed in bacteria (8.5%). Only subtypes 1, 3 and 5 were represented in the 111 archaeal genomes analyzed by the present inventors. However, the absence of subtypes 2, 4 and 6 from archaeal genomes could be the result of their rarity and the relative paucity of sequenced archaeal genomes, comprising only 111 out of the 1447 genomes analyzed.

Taken together, the BREX systems undergo extensive horizontal transfer, with subtype 1 possibly the ancestral form of BREX.

Example 6 Frequent Interruptions in the Adenine-Specific Methylase PglX

One of the type 1 BREX-containing Bacillus subtilis strains obtained was not active against any of the tested phages although PCR analysis showed that it contained the complete BREX system. Upon Illumina whole-genome re-sequencing of the engineered strain, a frameshift mutation in the adenine-specific methylase gene pglX was observed, resulting from a single nucleotide deletion occurring in a stretch of seven guanine (G) residues at position 2128 (out of 3539 bp) of this gene. These results further support that the pglX gene is essential for the function of the type 1 BREX system. Therefore more broadly additional evidence for genetic variability of pglX in nature was examined.

In 11% (15/135) of the BREX systems that were documented, the pglX gene presented irregularities with respect to the common BREX organization (FIG. 17A). These included seven instances of premature stop codons in the middle of the gene, two instances of gene duplication and six occurrences where a full length pglX gene was adjacent to one or more partial forms of pglX (with an extreme example in Methacobrevibacter smithii, where five truncated forms of the pglX are found near the full length gene (FIG. 17B). The complete and truncated forms of the methylase usually resided on opposite strands and were accompanied by a gene annotated as a recombinase, possibly involved in switching between the two versions of pglX. Indeed, when analyzing the genomes of two strains of Lactobacillus rhamnosus GG that were sequenced independently (NCBI accessions FM179322 (NC_013198 SEQ ID NO: 1) and AP011548 (NC_017482 SEQ ID NO: 2), the present inventors found that the pglX sequence was identical between the strains except for a cassette of 313 bp that was switched between the full length and truncated pglX genes (FIG. 17C). The interchanged cassette was flanked by two inverted repeats suggesting a recombination-based cassette switching possibly mediated by the accompanying recombinase.

DNA shuffling via recombination events was previously shown to control phase variation in bacterial defense-related genes to alter the specificity or to mitigate toxic effects of specific genes in the absence of phage pressure⁴⁻²⁶. Taken together, since no other gene except for pglX presented such high rates of irregularities, these results marked pglX as possibly undergoing frequent phase-variation, suggesting that this gene might confer specificity in the BREX system, or, alternatively, is particularly toxic.

Example 7 Extensive Identification of BREX Systems in Prokaryotic Genomes

Following the initial homology searches in 1447 genomes described in details hereinabove, the present inventors performed an extensive homology search on a bigger set of genomes, 2263 complete and 5493 draft genomes, using the 14 genes associated with BREX systems (Table 1 above). Only gene clusters containing the two core genes (pglX and brxC/pglY) and at least two additional genes were considered (Tables 10-16 above).

The homology searches of the BREX genes in the 5493 genomes found 536 BREX systems in 9.3% (513/5493) of all genomes analyzed.

BREX type 1, the most common form of BREX, appeared 409 times in 398 genomes (Tables 10 and 16 above).

In most cases a single BREX system per organism was found, with only 21 (4%) of genomes harboring more than one subtype (Table 16 above).

In addition, in 25% (134/536) of the identified systems, one of the genes was either missing or has become a pseudogene (Tables 10-15 above), possibly representing inactivated systems.

Furthermore, in 11.5% (62/536) of the BREX systems that were documented, the pglX gene presented irregularities with respect to the common BREX organization.

Taken together, the broader analysis of the 7756 genomes reinforced all findings obtained with the 1447 set of genomes described hereinabove.

Taken together, the above results described a phage resistance system widespread in bacteria and archaea, which the present inventors denoted BREX system. The BREX family of systems can be divided into six coherent subtypes containing 4-8 genes each, two of which are core genes, pglZ and brxC/pglY, present in all systems. The results also suggested pglX might confer specificity in the BREX system, or, alternatively, is particularly toxic. Moreover, the BREX systems undergo extensive horizontal transfer, with subtype 1, the most frequent subtype of this system, possibly the ancestral form of BREX.

In addition, the results demonstrated that the BREX type 1 system confers complete or partial resistance against phages spanning a wide phylogeny of phage types, including lytic and temperate phages, even in the first cycle of infection. The abundance of this system and the efficiency in which it protects against phages implies that it plays an important role as a major line of defense encoded by bacteria against phages.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

TABLE 17 SEQ ID NO. vs. Accession Number Index SEQ ID NO. Accession No. 3 643625571 4 640726035 5 640526647 6 643706992 7 649671139 8 378443454 9 644886774 10 438000910 11 2506688719 12 2506688721 13 643706994 14 326402148 15 344198243 16 169786889 17 407698262 18 86156430 19 56475432 20 146351220 21 374998023 22 386866198 23 410470815 24 323524377 25 330814956 26 134294128 27 313671969 28 78042616 29 189499000 30 374294493 31 374294493 32 300853232 33 302384444 34 302384444 35 339441064 36 339324158 37 257057919 38 270307451 39 300087139 40 89892746 41 408417460 42 256827818 43 402570638 44 239904639 45 46562128 46 387151873 47 297567992 48 401761514 49 385785459 50 387869382 51 259906682 52 85372828 53 386632422 54 386627502 55 157159467 56 260866153 57 218561636 58 172056045 59 347534971 60 302877245 61 302877245 62 239825584 63 400756305 64 332661890 65 386712343 66 435852812 67 397655102 68 385816611 69 301065125 70 385812838 71 403514032 72 268318562 73 338202359 74 385826720 75 258506995 76 296110131 77 148642060 78 397779166 79 409187964 80 435850242 81 20088899 82 21226102 83 88601322 84 88601322 85 336115651 86 83588874 87 304319677 88 403056439 89 118578449 90 194335182 91 91790731 92 330806657 93 392419087 94 93004831 95 226303489 96 192288433 97 90019649 98 378448274 99 383494824 100 16763390 101 378697983 102 379699217 103 378982542 104 378987404 105 330837866 106 117918459 107 113968346 108 257062754 109 284034943 110 313681130 111 427711179 112 114565576 113 114565576 114 85857845 115 438000910 116 332798023 117 237653092 118 409131816 119 304315537 120 390948458 121 431932943 122 229606122 123 360034408 124 260752245 125 83309099 126 83309099 127 120552944 128 186680550 129 338209545 130 257051090 131 257051090 132 169237353 133 169237353 134 344209485 135 344209485 136 345006827 137 345006827 138 433593057 139 433593057 140 296120274 141 220915123 142 262193326 143 262193326 144 302390797 145 117927211 146 392406391 147 150002608 148 312792283 149 219846956 150 317151727 151 146328629 152 289191496 153 289191496 154 336475959 155 189218017 156 83588874 157 77163561 158 300112745 159 154250456 160 154250456 161 325106586 162 297616214 163 297616214 164 332798023 165 269791619 166 320114857 167 289577265 168 167036431 169 390933132 170 433653743 171 333895862 172 83718394 173 257091663 174 383452024 175 383452024 176 340792956 177 86738724 178 312193897 179 262193326 180 262193326 181 336115651 182 302864508 183 145220606 184 379706264 185 121582711 186 257054089 187 134096620 188 433601838 189 430741030 190 162448269 191 32141095 192 182433793 193 72160406 194 296267998 195 83642913 196 432327926 197 392406391 198 169830219 199 206895078 200 428771848 201 428771848 202 291285947 203 431792069 204 253698656 205 327312315 206 148269145 207 325106586 208 83718394 209 257091663 210 383452024 211 340792956 212 86738724 213 312193897 214 262193326 215 336115651 216 302864508 217 145220606 218 379706264 219 121582711 220 257054089 221 134096620 222 433601838 223 430741030 224 162448269 225 162448269 226 162448269 227 32141095 228 182433793 229 72160406 230 296267998 231 83642913 232 396584758 233 452732384 234 472443541 235 336440820 236 476380507 237 374627927 238 424979852 239 472222746 240 419173668 241 331640257 242 375004550 243 421082290 244 417605122 245 332655353 246 373485774 247 417268567 248 363900611 249 419280801 250 262371200 251 256402721 252 262374339 253 418024796 254 472199351 255 419235449 256 352101099 257 226597601 258 455877139 259 407801688 260 424026168 261 443537933 262 329896015 263 433118449 264 313151045 265 470894026 266 420093456 267 443474995 268 423730148 269 452301294 270 452281584 271 320096521 272 373114969 273 421857666 274 422905795 275 421735395 276 293373625 277 424001199 278 410105720 279 301030692 280 319430354 281 472217717 282 423286106 283 425019536 284 238756169 285 398800292 286 433099189 287 452722814 288 419389168 289 242362072 290 422028620 291 442594974 292 407974869 293 476381541 294 452704090 295 452743095 296 425034026 297 254426770 298 424005355 299 418354477 300 450256199 301 223955923 302 419919231 303 452722284 304 449053971 305 386289604 306 359780050 307 411116154 308 224581172 309 314953942 310 452301765 311 422033758 312 422912392 313 296328579 314 294784664 315 262377679 316 418013522 317 476381295 318 293611286 319 423144195 320 224581037 321 469923807 322 336420422 323 237666990 324 417550237 325 390606126 326 254692771 327 211594571 328 415823418 329 301064104 330 425024171 331 238694717 332 386814329 333 476381563 334 295396063 335 419200062 336 423718272 337 423926641 338 424977968 339 424971161 340 229509077 341 340358330 342 424023365 343 424612424 344 443530314 345 424605785 346 357040530 347 455967306 348 425047878 349 149189552 350 385881638 351 427597607 352 389575461 353 442610050 354 182624174 355 314994688 356 211595724 357 419887425 358 472214599 359 422901577 360 406351917 361 225001047 362 419281346 363 356907979 364 419206516 365 406839856 366 440708760 367 424651801 368 472210693 369 196250231 370 476380904 371 427557910 372 224515124 373 381395491 374 422702452 375 409167416 376 476380862 377 358069018 378 421345543 379 417812631 380 401676751 381 314998443 382 421334477 383 423532168 384 314940712 385 149113251 386 422924875 387 423891867 388 419378419 389 427682108 390 423480222 391 421772441 392 448689403 393 421768382 394 427621994 395 343503287 396 472166833 397 418348058 398 427575979 399 419252283 400 251833313 401 455819031 402 424609621 403 476380950 404 469924283 405 472149938 406 417209278 407 390169096 408 455593728 409 424644158 410 452702507 411 399579771 412 472157757 413 224581092 414 427658715 415 424974911 416 417589425 417 294807000 418 419362951 419 419362951 420 345651578 421 373849145 422 410618100 423 224485776 424 298384183 425 298384183 426 419358064 427 419358064 428 419352509 429 419352509 430 417223690 431 417223690 432 381157709 433 282900768 434 425444918 435 472183758 436 424655749 437 334126448 438 126665966 439 423709466 440 437842748 441 418008622 442 469924241 443 410637153 444 363898451 445 472152144 446 419240736 447 419383938 448 424630728 449 374627936 450 374627936 451 418342894 452 420088308 453 255743719 454 455929923 455 206975561 456 423164021 457 417522340 458 419831962 459 139439021 460 314944229 461 423147095 462 224485442 463 422305960 464 225363921 465 417815500 466 425040990 467 343177620 468 423878932 469 211593993 470 418398711 471 378759082 472 402296507 473 259048327 474 455905617 475 224514842 476 357631901 477 384096966 478 425069205 479 265756999 480 427646214 481 403955718 482 373471569 483 456039135 484 224993695 485 149109670 486 365096837 487 298527635 488 395208219 489 458920171 490 419246266 491 423155496 492 421624890 493 427425534 494 458913812 495 417191381 496 424621178 497 262405036 498 422890700 499 419229938 500 424015456 501 335043876 502 419894708 503 472202862 504 211594035 505 424618529 506 224581212 507 169343945 508 405982616 509 469924367 510 417292897 511 418336054 512 423152690 513 419841668 514 421338374 515 325917999 516 365822320 517 294645413 518 294645413 519 419367985 520 419367985 521 419343475 522 419343475 523 365833706 524 365833706 525 448302553 526 448302553 527 448681954 528 448681954 529 448413196 530 448413196 531 470888868 532 443475057 533 386810750 534 196035064 535 419725778 536 336430981 537 242355593 538 397905651 539 390993910 540 323701113 541 288572734 542 325672510 543 408373871 544 423394306 545 254434980 546 334130722 547 472439485 548 398888999 549 196044104 550 374534854 551 415887008 552 343509531 553 381156824 554 374623705 555 326204471 556 326389902 557 256752440 558 256752440 559 211606481 560 384563951 561 453074660 562 375098335 563 359765453 564 451335404 565 126666487 566 257461537 567 440700728 568 163804182 569 458780588 570 383824531 571 224581088 572 383827549 573 365878943 574 254173939 575 149176214 576 458859600 577 441515884 578 163719735 579 381168746 580 211606481 581 288920043 582 452746574 583 319947885 584 2530355709 585 [Vibrio cholerae O1 str. 3582-05] 586 650306799 587 373108119 588 373108120 589 2520346679 590 384563951 591 453074660 592 375098335 593 359765453 594 451335404 595 126666487 596 257461537 597 440700728 598 163804182 599 458780588 600 383824531 601 224581088 602 383827549 603 365878943 604 254173939 605 149176214 606 458859600 607 441515884 608 163719735 609 381168746 610 211606481 611 211606481 612 288920043 613 452746574 614 643625571 615 640726035 616 640526647 617 326402593 618 344200467 619 169786942 620 407700180 621 86159654 622 56478399 623 403571624 624 374998147 625 386867049 626 410471296 627 323527069 628 330816348 629 134296119 630 313673973 631 78043274 632 189500610 633 374294726 634 374297213 635 300856435 636 302387137 637 302388015 638 310658340 639 339442903 640 339327420 641 257060038 642 270307693 643 300088703 644 89893422 645 408420313 646 256829113 647 402570961 648 239908251 649 46580432 650 387153155 651 297570109 652 401762042 653 385787389 654 387872412 655 259909432 656 85375773 657 386637212 658 386632292 659 157159791 660 260871024 661 218561659 662 172056357 663 347535921 664 302878116 665 302878741 666 239826789 667 39997206 668 332661908 669 386716367 670 435853826 671 397655646 672 385817606 673 301067094 674 385813807 675 403515035 676 268319508 677 338203660 678 385828737 679 258509093 680 296110179 681 148643811 682 397780111 683 410669357 684 435851550 685 20091205 686 21226256 687 88602373 688 88603150 689 336119448 690 83591063 691 304320702 692 403059708 693 118580704 694 194337068 695 253987912 696 91790843 697 330807434 698 392420329 699 93006433 700 226306383 701 192289536 702 90022402 703 378453461 704 383499055 705 16767740 706 378702329 707 379703733 708 378987163 709 378447803 710 378991757 711 330838575 712 117920265 713 113970047 714 257063041 715 284039691 716 313682811 717 427712030 718 114566521 719 114568000 720 85858751 721 438001502 722 332798529 723 217968746 724 410668521 725 304316006 726 390949848 727 431933022 728 229608798 729 360034503 730 260753605 731 83311139 732 83311142 733 120553494 734 186685708 735 338213689 736 336252376 737 336252378 738 257052978 739 257052980 740 169237555 741 169237557 742 222476093 743 222476095 744 344209884 745 344209886 746 345007036 747 345007038 748 433593287 749 433593289 750 296123320 751 220916262 752 262193921 753 262194480 754 302392144 755 117928020 756 392408179 757 150006230 758 312792857 759 219848033 760 317153364 761 146329396 762 289192375 763 289192395 764 336477235 765 189218341 766 83589500 767 77163603 768 300112782 769 154251634 770 154253986 771 325107712 772 297617452 773 297617463 774 438003073 775 332799809 776 269793099 777 320115755 778 289578545 779 167037339 780 390935377 781 433655486 782 333896806 783 83719623 784 257094926 785 383457202 786 383457204 787 340794637 788 86741641 789 312199460 790 262194454 791 262194456 792 336118509 793 302865796 794 145223789 795 379708000 796 121582862 797 257054591 798 134101639 799 433603653 800 430742881 801 162455964 802 21224932 803 182435399 804 72161113 805 296269522 806 83646203 807 432329234 808 392407895 809 169830968 810 206896068 811 428774400 812 428774405 813 291286510 814 431792782 815 253699434 816 327312662 817 148270869 818 396584764 819 458152047 820 472443547 821 336440365 822 432379520 823 374625979 824 424979861 825 472222755 826 419173682 827 331001731 828 375004439 829 421082306 830 417605263 831 332655414 832 373485787 833 417269258 834 363899395 835 419281128 836 262371247 837 260887939 838 262374465 839 418024979 840 472199360 841 419235503 842 352101112 843 229817501 844 443510724 845 407801756 846 424026177 847 443537945 848 329896082 849 433118460 850 307287300 851 470894102 852 420093469 853 443475020 854 423730158 855 458790583 856 458059910 857 320095081 858 373113625 859 421857673 860 422905804 861 421735408 862 293373689 863 424001208 864 410103029 865 301024307 866 317501018 867 472217726 868 423288074 869 425019539 870 238756191 871 398800471 872 433099194 873 457927366 874 419389186 875 229814945 876 422028737 877 442594980 878 407974988 879 432983127 880 457996554 881 458890354 882 425034029 883 254428479 884 424005364 885 418354555 886 450256209 887 313140119 888 419919260 889 458765352 890 449054087 891 386288258 892 359780190 893 366164460 894 317058268 895 314950595 896 458013691 897 422033788 898 422912400 899 296328627 900 294785588 901 262377713 902 418013529 903 432708023 904 293611306 905 423144203 906 223986438 907 430834455 908 336418208 909 237667881 910 417550828 911 388258093 912 257438026 913 114706331 914 415823688 915 301064167 916 425024180 917 227889976 918 386818311 919 432993617 920 295396111 921 419200149 922 423719993 923 423926650 924 424977971 925 424971164 926 229509126 927 340357065 928 424023373 929 424612433 930 443530596 931 424605794 932 357040625 933 443522967 934 425047890 935 149189596 936 386775208 937 427597640 938 389577851 939 442610063 940 182624228 941 314992449 942 153821425 943 419887467 944 472214607 945 422901580 946 404368577 947 86139886 948 419281501 949 357010162 950 419206641 951 407366555 952 440708892 953 424651810 954 472210701 955 196250357 956 432551902 957 427557941 958 210635261 959 381395616 960 422702473 961 409203050 962 432531965 963 358066714 964 421345612 965 417812642 966 401676815 967 314996685 968 421334490 969 423535444 970 314939179 971 167991284 972 422924884 973 423891876 974 419378582 975 427682140 976 423480949 977 421772573 978 448689857 979 421768511 980 427622024 981 343503399 982 472166842 983 418348068 984 427576009 985 419252317 986 227544817 987 443506619 988 424609629 989 432566736 990 431640910 991 472149947 992 417209350 993 390169177 994 443502704 995 424644167 996 458688573 997 404330919 998 472157766 999 302520233 1000 427658745 1001 424974920 1002 417589501 1003 294807071 1004 419363098 1005 419363099 1006 345508160 1007 373849352 1008 410618146 1009 224023322 1010 298384281 1011 298384294 1012 419358136 1013 419358137 1014 419352607 1015 419352608 1016 417224010 1017 417225258 1018 381159246 1019 282900778 1020 425444980 1021 472183766 1022 424655758 1023 334125671 1024 126665995 1025 423709629 1026 437842751 1027 418008631 1028 431534713 1029 410637164 1030 363897443 1031 472152152 1032 419240871 1033 419383944 1034 424630790 1035 374629429 1036 374630018 1037 418342902 1038 420088375 1039 255743727 1040 443518100 1041 206975617 1042 423164024 1043 417522346 1044 419832016 1045 139439065 1046 314941481 1047 423147141 1048 255014565 1049 422306080 1050 254850928 1051 417815511 1052 425040993 1053 340752704 1054 423878986 1055 90418997 1056 418398870 1057 381199747 1058 402700381 1059 260101568 1060 443514286 1061 160944224 1062 358012808 1063 383114787 1064 425069876 1065 265757019 1066 427646244 1067 404399332 1068 373469124 1069 443534358 1070 225374526 1071 167551250 1072 365096843 1073 298528556 1074 395208610 1075 458920176 1076 419246582 1077 423155504 1078 421624973 1079 427425586 1080 458913829 1081 417191550 1082 424621187 1083 262405137 1084 422890703 1085 419230121 1086 424015510 1087 335043947 1088 419894757 1089 472202871 1090 89073847 1091 424618575 1092 237744599 1093 169343946 1094 405982586 1095 431753901 1096 417292957 1097 418336066 1098 423152698 1099 419841735 1100 421338422 1101 325918007 1102 366087502 1103 294645433 1104 294645434 1105 419368080 1106 419368151 1107 419343546 1108 419343547 1109 365831806 1110 365831811 1111 448302568 1112 448302570 1113 448682078 1114 448682080 1115 448413236 1116 448413238 1117 470888966 1118 443475072 1119 386811046 1120 196035162 1121 419725844 1122 336426892 1123 257885934 1124 397905666 1125 392538885 1126 323701234 1127 288575102 1128 325266723 1129 408373945 1130 423398559 1131 254435628 1132 334130736 1133 472439490 1134 398889059 1135 196044278 1136 374812241 1137 415887047 1138 343509540 1139 381156866 1140 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375098335 2741 359765453 2742 451335404 2743 126666487 2744 257461537 2745 440700728 2746 163804182 2747 458780588 2748 383824531 2749 224581088 2750 383827549 2751 365878943 2752 254173939 2753 149176214 2754 458859600 2755 441515884 2756 163719735 2757 381168746 2758 211606481 2759 288920043 2760 319947885 2761 2530355711 2762 gi [Vibrio cholerae O1 str. 3582-05] 2763 650306800 2764 373108119 2765 373108120 2766 326402591 2767 326402592 2768 344200468 2769 344200470 2770 169786941 2771 407700179 2772 86159653 2773 56478402 2774 403571623 2775 374998146 2776 386867050 2777 323527068 2778 330816349 2779 313673970 2780 313673972 2781 78045163 2782 189500607 2783 374294727 2784 374297205 2785 374297212 2786 300856434 2787 302387135 2788 302388014 2789 302388025 2790 310658341 2791 339442905 2792 339327417 2793 257060035 2794 257060036 2795 270307692 2796 300088702 2797 89893423 2798 408420312 2799 256829112 2800 402570962 2801 239908250 2802 46580429 2803 387153158 2804 297570107 2805 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REFERENCES Other References are Cited in the Application

-   1. Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage     resistance mechanisms. Nature reviews. Microbiology 8, 317-327     (2010). -   2. Stern, A. & Sorek, R. The phage-host arms race: shaping the     evolution of microbes. Bioessays 33, 43-51 (2011). -   3. Tock, M. R. & Dryden, D. T. The biology of restriction and     anti-restriction. Curr Opin Microbiol 8, 466-472 (2005). -   4. Chopin, M. C., Chopin, A. & Bidnenko, E. Phage abortive infection     in lactococci: variations on a theme. Curr Opin Microbiol 8, 473-479     (2005). -   5. Deveau, H., Garneau, J. E. & Moineau, S. CRISPR/Cas system and     its role in phage-bacteria interactions. Annu Rev Microbiol 64,     475-493 (2010). -   6. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of     bacteria and archaea. Science 327, 167-170 (2010). -   7. Sorek. R., Kunin, V. & Hugenholtz, P. CRISPR—a widespread system     that provides acquired resistance against phages in bacteria and     archaea. Nature reviews. Microbiology 6, 181-186 (2008). -   8. van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M. &     Brouns. S. J. CRISPR-based adaptive and heritable immunity in     prokaryotes. Trends Biochem Sci 34, 401-407 (2009). -   9. Makarova, K. S., Wolf, Y. I. & Koonin, E. V. Comparative genomics     of defense systems in archaea and bacteria. Nucleic acids research     41, 4360-4377 (2013). -   10. Chinenova. T. A., Mkrtumian, N. M. & Lomovskaia, N. D. [Genetic     characteristics of a new phage resistance trait in Streptomyces     coelicolor A3(2)]. Genetika 18, 1945-1952 (1982). -   11. Sumby, P. & Smith, M. C. Genetics of the phage growth limitation     (Pgl) system of Streptomyces coelicolor A3(2). Mol Microbiol 44,     489-500 (2002). -   12. Laity, C., Chater, K. F., Lewis, C. G. & Buttner. M. J. Genetic     analysis of the phi C31-specific phage growth limitation (Pgl)     system of Streptomyces coelicolor A3(2). Mol Microbiol 7, 329-336     (1993). -   13. Makarova, K. S., Wolf, Y. I., Snir, S. & Koonin, E. V. Defense     islands in bacterial and archaeal genomes and prediction of novel     defense systems. J Bacteriol 193, 6039-6056 (2011). -   14. Rudinski, M. S. & Dean, D. H. Evolutionary considerations of     related Bacillus subtilis temperate phages phi 105, rho 14, rho 10,     and rho 6 as revealed by heteroduplex analysis. Virology 99, 57-65     (1979). -   15. Bondy-Denomy, J., Pawluk, A., Maxwell, K. L. & Davidson. A. R.     Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune     system. Nature 493, 429-432 (2013). -   16. Wang, L. et al. DNA phosphorothioation is widespread and     quantized in bacterial genomes. Proc Natl Acad Sci USA 108,     2963-2968 (2011). -   17. You, D., Wang, L., Yao, F., Zhou, X. & Deng, Z. A novel DNA     modification by sulfur: DndA is a NifS-like cysteine desulfurase     capable of assembling DndC as an iron-sulfur cluster protein in     Streptomyces lividans. Biochemistry 46, 6126-6133 (2007). -   18. Wang, L. et al. Phosphorothioation of DNA in bacteria by dnd     genes. Nat Chem Biol 3, 709-710 (2007). -   19. Gomez, P. & Buckling, A. Bacteria-phage antagonistic coevolution     in soil. Science 332, 106-109 (2011). -   20. Hall, A. R., Scanlan, P. D., Morgan, A. D. & Buckling, A.     Host-parasite coevolutionary arms races give way to fluctuating     selection. Ecol Lett 14, 635-642 (2011). -   21. Makarova, K. S., Anantharaman, V., Aravind, L. & Koonin, E. V.     Live virus-free or die: coupling of antivirus immunity and     programmed suicide or dormancy in prokaryotes. Biol Direct 7, 40     (2012). -   22. Makarova, K. S. et al. Evolution and classification of the     CRISPR-Cas systems. Nature reviews. Microbiology 9, 467-477 (2011). -   23. Sumby, P. & Smith, M. C. Phase variation in the phage growth     limitation system of Streptomyces coelicolor A3(2). J Bacteriol 185,     4558-4563 (2003). -   24. Bikard, D. & Marraffini, L. A. Innate and adaptive immunity in     bacteria: mechanisms of programmed genetic variation to fight     bacteriophages. Curr Opin Immunol 24, 15-20 (2012). -   25. Hallet, B. Playing Dr Jekyll and Mr Hyde: combined mechanisms of     phase variation in bacteria. Curr Opin Microbiol 4, 570-581 (2001). -   26. Cerdeno-Tarraga, A. M. et al. Extensive DNA inversions in the B.     fragilis genome control variable gene expression. Science 307,     1463-1465 (2005). -   27. Terns, M. P. & Terns, R. M. CRISPR-based adaptive immune     systems. Curr Opin Microbiol 14, 321-327 (2011). -   28. Haft, D. H., Selengut. J., Mongodin, E. F. & Nelson, K. E. A     guild of 45 CRISPR-associated (Cas) protein families and multiple     CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol     1, e60 (2005). -   29. Stern, A., Keren. L., Wurtzel. O., Amitai, G. & Sorek, R.     Self-targeting by CRISPR: gene regulation or autoimmunity? Trends     Genet 26, 335-340 (2010). -   30. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas     systems. Science 339, 819-823 (2013). -   31. DiCarlo. J. E. et al. Genome engineering in Saccharomyces     cerevisiae using CRISPR-Cas systems. Nucleic acids research 41,     4336-4343 (2013). -   32. Suchard. M. A. & Redelings. B. D. BAli-Phy: simultaneous     Bayesian inference of alignment and phylogeny. Bioinformatics 22,     2047-2048 (2006). -   33. Loytynoja, A. & Goldman, N. Phylogeny-aware gap placement     prevents errors in sequence alignment and evolutionary analysis.     Science 320, 1632-1635 (2008). -   34. Marchler-Bauer, A. & Bryant, S. H. CD-Search: protein domain     annotations on the fly. Nucleic acids research 32. W327-331 (2004). -   35. Soding, J. Protein homology detection by HMM-HMM comparison.     Bioinformatics 21, 951-960 (2005). 

1. A nucleic acid construct comprising a polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW; a polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; or a polynucleotide comprising a nucleic acid sequence encoding a BREX system comprising: (i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL; (ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII; (iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI; (iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA; (v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI; or (vi) brxP, brxC/pglY, pglZ and brx, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same. 2-4. (canceled)
 5. A nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW.
 6. A nucleic acid construct system comprising at least two nucleic acid constructs expressing a BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
 7. A phage defense composition, comprising as an active ingredient a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; and an acceptable carrier or diluent.
 8. (canceled)
 9. An isolated cell genetically modified to express a BREX system selected from the group consisting of (1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, (2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, (3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL, (3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII, (3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI, (3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA, (3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or (3vi) brxP, brxC/pglY, pglZ and brxL. 10-13. (canceled)
 14. An isolated cell genetically modified to express a BREX system polypeptide selected from the group consisting of pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.
 15. (canceled)
 16. A method of protecting bacteria from phage attack, the method comprising expressing in the bacteria the polynucleotide of claim 1, thereby protecting the bacteria from phage attack.
 17. A method of protecting first bacteria from phage attack, the method comprising contacting the first bacteria with second bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHI, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, wherein the first bacteria and said second bacteria are non identical; thereby protecting the bacteria from phage attack.
 18. The method of claim 17 wherein said first bacteria does not express a BREX system endogenously.
 19. The method of claim 16 wherein the bacteria does not express a BREX system endogenously.
 20. The method of claim 16, wherein said phage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T and SPO2.
 21. The method of claim 16, wherein said phage is not Φ105, rho10 and rho14.
 22. The method of claim 16 wherein said phage is a lytic phage.
 23. (canceled)
 24. An isolated bacteria comprising a nucleic acid sequence encoding a BREX system and a transmissible genetic element expressing said BREX system, wherein said isolated bacteria do not endogenously express said BREX system and wherein said BREX system comprises brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, or comprises brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.
 25. The method of claim 16, wherein said BREX system is type 1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.
 26. The method of claim 16, wherein: (i) brxC/pglY is selected from the group of SEQ ID NO: 3-155, 157-765 and 767-1175, (ii) pglZ is selected from the group of SEQ ID NO: 1176-1318, 1320-1856, 1858-2250, 6204 and 6205, (iii) pglX is selected from the group of SEQ ID NO: 2251-3280 and 6186-6201, (iv) pglXI is selected from the group of SEQ ID NO: 3281-3296, 3298-3356 and 3358-3403, (v) brxP is selected from the group of SEQ ID NO: 3404-3440, (vi) brxHI is selected from the group of SEQ ID NO: 3543-3642, (vii) brxHII is selected from the group of SEQ ID NO: 3441-3460, 3462-3511 3513-3542 and 6173-6185, (viii) brxL is selected from the group of SEQ ID NO: 3643-4412, 6165, 6166, 6169, 6170, 6202 and 6203, (ix) brxD is selected from the group of SEQ ID NO: 4413-4488, (x) brxA is selected from the group of SEQ ID NO: 4489-4621, 4623-5086, 5088-5415, 6167, 6168, 6171 and 6172, (xi) brxB is selected from the group of SEQ ID NO: 5416-5947, 6206-6209, (xii) brxF is selected from the group of SEQ ID NO: 5948-5957, 5959-5988 and 5990-6028, (xiii) brxE is selected from the group of SEQ ID NO: 6029-6040 and (xiv) pglW is selected from the group of SEQ ID NO: 6041-6138.
 27. A method of inducing phage sensitivity in a bacterial cell, the method comprising contacting a bacterial cell which expresses a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, or comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducing sensitivity of the bacterial cell to phage infection.
 28. An isolated bacteria generated according to the method of claim
 27. 29. (canceled)
 30. A method for preparing a food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product comprising adding to said food, food additive, feed, nutritional supplement, probiotic supplement, a personal care product, a health care product, and a veterinary product a bacteria which expresses on a transmissible genetic element a BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, or comprising brxC/pglY, pglZ pglX, pglW and at least one of brxD and brxHI, thereby preparing the food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, and veterinary product.
 31. (canceled)
 32. The method of claim 30, wherein said food or feed is a dairy product. 33-38. (canceled)
 39. A method of treating a microbial infection in a subject in need thereof, the method comprising contacting the bacteria with an anti BREX system agent capable of down regulating a BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby treating the infection.
 40. The method of claim 39 further comprising administering to the subject a phage therapy and/or an antibiotic. 41-50. (canceled)
 51. The method of claim 17, wherein said phage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T and SPO2.
 52. The method of claim 17, wherein said phage is not Φ105, rho10 and rho14.
 53. The method of claim 17, wherein said phage is a lytic phage.
 54. The method of claim 17, wherein said BREX system is type 1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.
 55. The method of claim 17, wherein: (xv) brxC/pglY is selected from the group of SEQ ID NO: 3-155, 157-765 and 767-1175, (xvi) pglZ is selected from the group of SEQ ID NO: 1176-1318, 1320-1856, 1858-2250, 6204 and 6205, (xvii) pglX is selected from the group of SEQ ID NO: 2251-3280 and 6186-6201, (xviii) pglXI is selected from the group of SEQ ID NO: 3281-3296, 3298-3356 and 3358-3403, (xix) brxP is selected from the group of SEQ ID NO: 3404-3440, (xx) brxHI is selected from the group of SEQ ID NO: 3543-3642, (xxi) brxHII is selected from the group of SEQ ID NO: 3441-3460, 3462-3511 3513-3542 and 6173-6185, (xxii) brxL is selected from the group of SEQ ID NO: 3643-4412, 6165, 6166, 6169, 6170, 6202 and 6203, (xxiii) brxD is selected from the group of SEQ ID NO: 4413-4488, (xxiv) brxA is selected from the group of SEQ ID NO: 4489-4621, 4623-5086, 5088-5415, 6167, 6168, 6171 and 6172, (xxv) brxB is selected from the group of SEQ ID NO: 5416-5947, 6206-6209, (xxvi) brxF is selected from the group of SEQ ID NO: 5948-5957, 5959-5988 and 5990-6028, (xxvii) brxE is selected from the group of SEQ ID NO: 6029-6040, and (xxviii) pglW is selected from the group of SEQ ID NO: 6041-6138. 